Antibodies against cd73 and uses thereof

ABSTRACT

The present invention provides isolated monoclonal antibodies, particularly human antibodies, that bind to human Cluster of Differentiation 73 (CD73) with high affinity, and inhibit the activity of CD73, and optionally mediate antibody dependent CD73 internalization. Nucleic acid molecules encoding the antibodies of the invention, expression vectors, host cells and methods for expressing the antibodies of the invention are also provided. Immunoconjugates, bispecific molecules and pharmaceutical compositions comprising the antibodies of the invention are also provided. The invention also provides methods for inhibiting the growth of a tumor cell expressing CD73 using the antibodies of the invention, including methods for treating various cancers.

This application is a continuation of U.S. patent application Ser. No.15/520,638 (allowed), entitled “Antibodies Against CD73 and UsesThereof” filed on Apr. 20, 2017, which is a 35 U.S.C. 371 national stagefiling of PCT Application No. PCT/US2015/061639, entitled “AntibodiesAgainst CD73 and Uses Thereof” filed Nov. 19, 2015, which claimspriority to U.S. Provisional Application No. 62/083,056, entitled“Antibodies Against CD73 and Uses Thereof” filed on Nov. 21, 2014, thecontents of each of which are hereby incorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 27, 2018 isnamed MXI-538USCN_Sequence_Listing.txt and is 608,870 bytes in size.

BACKGROUND

Cluster of Differentiation 73 (CD73), also known as ecto-5′-nucleotidase(ecto-5′NT, EC 3.1.3.5), is a glycosyl-phosphatidylinositol (GPI)-linkedcell surface enzyme found in most tissues, but particularly expressed inendothelial cells and subsets of hematopoietic cells (Resta et al.,Immunol Rev 1998; 161:95-109 and Colgan et al., Prinergic Signal 2006;2:351-60). CD73 is known to catalyze the dephosphorylation ofextracellular nucleoside monophosphates into nucleosides, such asadenosine. Adenosine is a widely studied signaling molecule whichmediates its biological effects through several receptors, including A1,A2A, A2B, and A3. Adenosine has been shown to regulate proliferation andmigration of many cancers and to have an immunosuppressive effectthrough the regulation of anti-tumor T cells (Zhang et al., Cancer Res2010; 70:6407-11).

CD73 has been reported to be expressed on many different cancers,including colon, lung, pancreas, ovary, bladder, leukemia, glioma,glioblastoma, melanoma, thyroid, esophageal, prostate and breast cancers(Jin et al., Cancer Res 2010; 70:2245-55 and Stagg et al., PNAS 2010;107:1547-52). Moreover, CD73 expression in cancer has been linked toincreased proliferation, migration, neovascularization, invasiveness,metastesis and shorter patient survival. CD73 activity has also beenproposed as a prognostic marker in papillary thyroid carcinomas. WhileCD73 has been shown to regulate cell-cell and cell-matrix interactionson tumor cells, CD73 expression and activity has also been linked toreduced T-cell responses and implicated in drug resistance (Spychala etal., Pharmacol Ther 3000; 87:161-73). Thus CD73 can regulate cancerprogression both directly and indirectly, which highlights its potentialas a novel therapeutic target.

Given the ongoing need for improved strategies for targeting diseasessuch as cancer, methods of regulating tumor progression through multiplemechanisms, as well as methods for regulating CD73 activity and relatedtherapeutic agents are highly desirable.

SUMMARY

Provided herein are isolated antibodies, such as monoclonal antibodies(antigen binding portions thereof), in particular human monoclonalantibodies, that specifically bind CD73 and have desirable functionalproperties. These properties include high affinity binding to humanCD73, binding to monkey CD73 (e.g., cynomolgus CD73), and the ability toinhibit CD73 enzymatic activity. The antibodies described herein can beused to inhibit tumor growth, reduce adenosine production, stimulate animmune response, and detect CD73 protein in a sample.

In certain embodiments, the anti-CD73 antibodies exhibit at least one ofthe following properties:

(a) inhibition of CD73 enzymatic activity;

(b) binding to cynomolgus CD73;

(c) antibody mediated internalization of CD73 into cells, e.g., tumorcells; and

(d) binding to a conformational epitope comprising amino acids 65-83 and157-172 of human CD73.

In certain embodiments, the anti-CD73 antibodies, or antigen bindingportions thereof, bind to human CD73 (monomeric and/or dimeric CD73)with a K_(D) of about 10 nM to 0.1 nM or less as measured, e.g., byBIACORE® SPR analysis, and is internalized into tumor cells with aT_(1/2) of no more than 10 min as measured, e.g., by pulse chase and asdescribed in the Examples.

In certain embodiments, the anti-CD73 antibodies, or antigen bindingportions thereof, bind within the regions FTKVQQIRRAEPNVLLLDA (SEQ IDNO: 96) and/or LYLPYKVLPVGDEVVG (SEQ ID NO: 97) of human CD73, e.g.,wherein the epitope spans or overlaps with FTKVQQIRRAEPNVLLLDA (SEQ IDNO: 96) and/or LYLPYKVLPVGDEVVG (SEQ ID NO: 97) of human CD73.

In certain embodiments, the anti-CD73 antibodies, or antigen bindingportions thereof, bind to an epitope on human CD73 (SEQ ID NO: 1) whichincludes all or a portion of amino acid residues FTKVQQIRRAEPNVLLLDA(SEQ ID NO: 96) and/or LYLPYKVLPVGDEVVG (SEQ ID NO: 97). In certainembodiments, the epitope bound by the anti-CD73 antibodies, or antigenbinding portions thereof, is determined by, e.g., HDX-MS and/orcrystallography.

In certain embodiments, the anti-CD73 antibodies comprise the threevariable heavy chain CDRs and the three variable light chain CDRs thatare in the variable heavy chain and variable light chain pairs ofanti-CD73 antibodies described herein, such as the chains in Table 35,e.g., SEQ ID NOs: 4 and 8; SEQ ID NOs: 4 and 12; SEQ ID NOs: 16 and 20;SEQ ID NOs: 16 and 24; SEQ ID NOs: 16 and 28; SEQ ID NOs: 32 and 36; SEQID NOs: 40 and 44; SEQ ID NOs: 40 and 48; SEQ ID NOs: 52 and 56; SEQ IDNOs: 60 and 64; SEQ ID NOs: 68 and 72; SEQ ID NOs: 68 and 76; SEQ IDNOs: 80 and 84; SEQ ID NOs: 88 and 92; SEQ ID NOs: 135 and 8; or SEQ IDNOs: 135 and 12. For example, the anti-CD73 antibodies comprise:

(a) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 5,6, and 7, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 9, 10, and 11, respectively;

(b) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 5,6, and 7, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 13, 14, and 15, respectively;

(c) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:17, 18, and 19, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 21, 22, and 23, respectively;

(d) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:17, 18, and 19, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 25, 26, and 27, respectively;

(e) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:17, 18, and 19, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 29, 30, and 31, respectively;

(f) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:33, 34, and 35, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 37, 38, and 39, respectively;

(g) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:41, 42, and 43, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 45, 46, and 47, respectively;

(h) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:41, 42, and 43, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 49, 50, and 51, respectively;

(i) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:53, 54, and 55, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 57, 58, and 59, respectively;

(j) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:61, 62, and 63, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 65, 66, and 67, respectively;

(k) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:69, 70, and 71, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 73, 74, and 75, respectively;

(l) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:69, 70, and 71, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 77, 78, and 79, respectively;

(m) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:81, 82, and 83, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 85, 86, and 87, respectively; or

(n) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:89, 90, and 91, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 93, 94, and 95, respectively.

In certain embodiments, the anti-CD73 antibodies, or antigen bindingportions thereof, comprise heavy chain CDR1, CDR2, and CDR3 sequences,SEQ ID NOs: 5, 6, and 7, respectively, and/or light chain CDR1, CDR2,and CDR3 sequences, SEQ ID NOs: 9, 10, and 11, respectively.

In certain embodiments, the anti-CD73 antibodies, or antigen bindingportions thereof, comprise heavy chain CDR1, CDR2, and CDR3 sequences,SEQ ID NOs: 5, 6, and 7, respectively, and/or light chain CDR1, CDR2,and CDR3 sequences, SEQ ID NOs: 13, 14, and 15, respectively.

In certain embodiments, the anti-CD73 antibodies, or antigen bindingportions thereof, comprise heavy and light chain variable regions havingamino acid sequences which are at least 80% identical, e.g., at least85% identical, at least 90% identical, at least 95% identical, at least96% identical, at least 97% identical, at least 98% identical, or atleast 99% identical or greater to the amino acid sequences of a heavychain variable region of anti-CD73 antibodies described herein, e.g.,comprising the amino acid sequence set forth in SEQ ID NOs: 4, 16, 32,40, 52, 60, 68, 80, 88, 135, 170-177 and/or a light chain variableregion of anti-CD73 antibodies described herein, e.g., comprising theamino acid sequence set forth in SEQ ID NOs: 8, 12, 20, 24, 28, 36, 44,48, 56, 64, 72, 76, 84, or 92.

In certain embodiments, the anti-CD73 antibodies, or antigen bindingportions thereof, comprise heavy chain and light chain variable regionsequences which are at least 80% identical, e.g., at least 85%identical, at least 90% identical, at least 95% identical, at least 96%identical, at least 97% identical, at least 98% identical, or at least99% identical or greater (e.g., 100%), to the amino acid sequences: SEQID NOs: 135 and 8; SEQ ID NOs: 135 and 12; SEQ ID NOs: 4 and 8; SEQ IDNOs: 4 and 12; SEQ ID NOs: 16 and 20; SEQ ID NOs: 16 and 24; SEQ ID NOs:16 and 28; SEQ ID NOs: 32 and 36; SEQ ID NOs: 40 and 44; SEQ ID NOs: 40and 48; SEQ ID NOs: 52 and 56; SEQ ID NOs: 60 and 64; SEQ ID NOs: 68 and72; SEQ ID NOs: 68 and 76; SEQ ID NOs: 80 and 84; SEQ ID NOs: 88 and 92;SEQ ID NO: 170 and any one of SEQ ID NOs: 20, 24 and 28; any one of SEQID NOs: 171-176 and SEQ ID NO: 8 or 12; or SEQ ID NO: 177 and 36.

In certain embodiments, the anti-CD73 antibodies, or antigen bindingportions thereof, comprise heavy and light chain variable regionsselected from the group consisting of: SEQ ID NOs: 135 and 8; SEQ IDNOs: 135 and 12; SEQ ID NOs: 4 and 8; SEQ ID NOs: 4 and 12; SEQ ID NOs:16 and 20; SEQ ID NOs: 16 and 24; SEQ ID NOs: 16 and 28; SEQ ID NOs: 32and 36; SEQ ID NOs: 40 and 44; SEQ ID NOs: 40 and 48; SEQ ID NOs: 52 and56; SEQ ID NOs: SEQ ID NOs: 60 and 64; SEQ ID NOs: 68 and 72; SEQ IDNOs: 68 and 76; SEQ ID NOs: 80 and 84; SEQ ID NOs: 88 and 92; SEQ ID NO:170 and any one of SEQ ID NOs: 20, 24 and 28; any one of SEQ ID NOs:171-176 and SEQ ID NO: 8 or 12; or SEQ ID NOs: 177 and 36.

In certain embodiments, the antibody or antigen binding portion thereof,comprises a heavy chain variable region having the amino acid sequenceset forth in SEQ ID NO: 135 and a light chain variable region having theamino acid sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 12.

Provided herein are anti-CD73 antibodies, or antigen binding portionsthereof, respectively, having full length heavy chain and light chainsequences at least 80%, 85%, 90%, 95%, 96%, 97%, 98% 99% or 100%identical to the amino acid sequences of any anti-CD73 antibodydescribed herein, e.g., SEQ ID NOs: 100 and 101; SEQ ID NOs: 100 and102; SEQ ID NOs: 103 and 104; SEQ ID NOs: 103 and 105; SEQ ID NOs: 103and 106; SEQ ID NOs: 107 and 108; SEQ ID NOs: 109 and 110; SEQ ID NOs:109 and 111; SEQ ID NOs: 112 and 113; SEQ ID NOs: 114 and 115; SEQ IDNOs: 116 and 117; SEQ ID NOs: 116 and 118; SEQ ID NOs: 119 and 120; SEQID NOs: 121 and 122; SEQ ID NOs: 133 or 189 (without C-terminal lysine)and 101; SEQ ID NOs: 133 or 189 and 102; SEQ ID NOs: 189 and 101; SEQ IDNOs: 189 and 102; any one of SEQ ID NOs: 184-186 and any one of SEQ IDNOs: 104-106; any one of SEQ ID NOs: 187-207 and SEQ ID NO: 101 or 102;or any one of SEQ ID NOs: 208-210 and SEQ ID NO: 108.

In certain embodiments, the antibody or antigen binding portion thereof,comprises a heavy chain region having the amino acid sequence set forthin SEQ ID NO: 133 or 189 and a light chain region having the amino acidsequence set forth in SEQ ID NO: 101 or SEQ ID NO: 102.

In certain embodiments, the anti-CD73 antibodies, or antigen-bindingportions thereof, are IgG1, IgG2, IgG3, or IgG4 antibodies, or variantsthereof. In certain embodiments, the anti-CD73 antibodies, or antigenbinding portions thereof, bind the same epitope on CD73 as theantibodies having the foregoing heavy and light chain variable regionssequences (e.g., antibodies CD73.4-1, CD73.4-2, CD73.3, 11F11-1,11F11-2, 4C3-1, 4C3-2, 4C3-3, 4D4, 10D2-1, 10D2-2, 11A6, 24H2, 5F8-1,5F8-2, 6E11 and/or 7A11).

In certain embodiments, the anti-CD73 antibodies, or antigen bindingportions thereof, comprise a modified heavy chain constant region whichincludes, e.g., a human CH1 domain, a human hinge domain, a human CH2domain, and a human CH3 domain in order from N- to C-terminus, whereinat least 2 domains are of a different isotype (e.g., IgG1, IgG2, IgG3,and IgG4 isotypes). For example, in certain embodiments, a modifiedconstant region comprises a human IgG2 hinge and at least one of theCH1, CH2, and CH3 domains is not of an IgG2 isotype. In certainembodiments, CH1 is a human IgG2 CH1 domain, e.g., having the amino acidsequence ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV (SEQ ID NO: 124). The modifiedconstant region may include a human IgG2 hinge domain, e.g., a humanIgG2 hinge domain which reduces heterogeneity in the cysteine binding,e.g., a human IgG2 hinge domain having amino acid substitution at C219,e.g., C219S, relative to a wildtype human IgG2 hinge domain (SEQ NO136), e.g., a human IgG2 hinge domain having the amino acid sequenceERKSCVECPPCPAPPVAG (SEQ ID NO: 123). The modified constant region mayinclude a human IgG1 CH2 domain which reduces or eliminates effectorfunctions, e.g., a human IgG1 CH2 domain having amino acid substitutionsA330S and P331S, relative to a wildtype human IgG1 CH2 domain (SEQ IDNO: 137), e.g., a human IgG1 CH2 domain comprises the amino acidsequence

(SEQ ID NO: 125) PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTIS KAK.The modified constant region may include a wildtype human IgG1 CH3domain, e.g., having the amino acid sequence

(SEQ ID NO: 128) GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK.

In certain embodiments, an anti-CD73 antibody comprises a modified heavychain region, e.g., comprising (i) an IgG2 hinge or (ii) an IgG2 hingeand IgG CH1 domain, with the proviso that the antibody is not an IgG2antibody, e.g., the antibody does not comprise a wildtype IgG2 heavychain constant region.

In certain embodiments, the anti-CD73 antibodies, or antigen bindingportions thereof, comprise any one of the constant regions describedherein, e.g., constant regions comprising the amino acid sequences setforth in SEQ ID NOs: 126, 127, 129, 130, 162-169, 180-183, 267-282, and300-347.

In certain embodiments, an anti-CD73 antibody comprises the VH and VLdomains of 11F11 or CD73.4, and a heavy chain constant region comprisingan IgG2 hinge or a heavy chain constant region comprising an IgG2 hingeand IgG2 CH1 domain. The heavy chain constant region may beIgG2CS-IgG1.1f (SEQ ID NO: 169) or IgG2CS-IgG1f (SEQ ID NO: 165),wherein “CS” refers to “C219S.”

Provided herein are bispecific molecules comprising the anti-CD73antibodies, or antigen binding portions thereof, linked to a moleculehaving a second binding specificity, as well as immunoconjugatescomprising the anti-CD73 antibodies, or antigen binding portionsthereof, linked to an agent.

Nucleic acid molecules encoding the heavy and/or light chain variableregion sequences of the anti-CD73 antibodies are also provided, as wellas expression vectors comprising the nucleic acid molecules, and cellstransformed with the expression vectors.

Compositions and kits comprising anti-CD73 antibodies, or antigenbinding portions thereof, are also provided.

Provided herein is a method of preparing anti-CD73 antibodies,comprising expressing an anti-CD73 antibody disclosed herein in a celland isolating the antibody from the cell.

Methods of using the anti-CD73 antibodies disclosed herein are alsoprovided, e.g., methods of decreasing adenosine levels, e.g., in or by atumor, e.g., a tumor cell, expressing CD73, methods of stimulating anantigen-specific T cell response, methods of stimulating an immuneresponse in a subject, methods of inhibiting the growth of tumor cellsin a subject, methods of treating cancer, e.g., by immunotherapy. Incertain embodiments, the cancer is bladder cancer, breast cancer,uterine/cervical cancer, ovarian cancer, prostate cancer, testicularcancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer,colorectal cancer, colon cancer, kidney cancer, head and neck cancer,lung cancer, stomach cancer, germ cell cancer, bone cancer, livercancer, thyroid cancer, skin cancer, neoplasm of the central nervoussystem, lymphoma, leukemia, myeloma, sarcoma, or virus-related cancer.The cancer may be a metastatic cancer, refractory cancer, or recurrentcancer.

In certain embodiments, the methods described herein further compriseadministering one or more additional therapeutics, e.g., a therapeuticthat stimulates the immune system, e.g., a PD-1 antagonist, a CTLA-4antagonist, a LAG-3 antagonist, a GITR antagonist, and/or an anti-CD39antibody, an anti-A2AR antibody, or chemical inhibitor of A2AR.

Other features and advantages of the instant disclosure will be apparentfrom the following detailed description and examples, which should notbe construed as limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the nucleotide sequence (SEQ ID NO: 237) and amino acidsequence (SEQ ID NO: 135) of the heavy chain variable region of theCD73.4-1 human monoclonal antibody. The CDR1 (SEQ ID NO: 5), CDR2 (SEQID NO: 6) and CDR3 (SEQ ID NO: 7) regions are delineated and the V, Dand J germline derivations are indicated.

FIG. 1B shows the nucleotide sequence (SEQ ID NO: 140) and amino acidsequence (SEQ ID NO: 8) of the light chain variable region (VK1) of theCD73.4-1 human monoclonal antibody. The CDR1 (SEQ ID NO: 9), CDR2 (SEQID NO: 10) and CDR3 (SEQ ID NO: 11) regions are delineated and the V, Dand J germline derivations are indicated.

FIG. 2A shows the nucleotide sequence (SEQ ID NO: 237) and amino acidsequence (SEQ ID NO: 135) of the heavy chain variable region of theCD73.4-2 human monoclonal antibody. The CDR1 (SEQ ID NO: 5), CDR2 (SEQID NO: 6) and CDR3 (SEQ ID NO: 7) regions are delineated and the V, Dand J germline derivations are indicated.

FIG. 2B shows the nucleotide sequence (SEQ ID NO: 141) and amino acidsequence (SEQ ID NO: 12) of the light chain variable region of theCD73.4-2 human monoclonal antibody. The CDR1 (SEQ ID NO: 13), CDR2 (SEQID NO: 14) and CDR3 (SEQ ID NO: 15) regions are delineated and the V, Dand J germline derivations are indicated.

FIG. 3A shows the nucleotide sequence (SEQ ID NO: 139) and amino acidsequence (SEQ ID NO: 4) of the heavy chain variable region of the11F11-1 human monoclonal antibody. The CDR1 (SEQ ID NO: 5), CDR2 (SEQ IDNO: 6) and CDR3 (SEQ ID NO: 7) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 3B shows the nucleotide sequence (SEQ ID NO: 140) and amino acidsequence (SEQ ID NO: 8) of the light chain variable region of the11F11-1 human monoclonal antibody. The CDR1 (SEQ ID NO: 9), CDR2 (SEQ IDNO: 10) and CDR3 (SEQ ID NO: 11) regions are delineated and the V, D andJ germline derivations are indicated.

FIG. 4A shows the nucleotide sequence (SEQ ID NO: 139) and amino acidsequence (SEQ ID NO: 4) of the heavy chain variable region of the11F11-2 human monoclonal antibody. The CDR1 (SEQ ID NO: 5), CDR2 (SEQ IDNO: 6) and CDR3 (SEQ ID NO: 7) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 4B shows the nucleotide sequence (SEQ ID NO: 141) and amino acidsequence (SEQ ID NO: 12) of the light chain variable region of the11F11-2 human monoclonal antibody. The CDR1 (SEQ ID NO: 13), CDR2 (SEQID NO: 14) and CDR3 (SEQ ID NO: 15) regions are delineated and the V, Dand J germline derivations are indicated.

FIG. 5A shows the nucleotide sequence (SEQ ID NO: 142) and amino acidsequence (SEQ ID NO: 16) of the heavy chain variable region of the 4C3-1human monoclonal antibody. The CDR1 (SEQ ID NO: 17), CDR2 (SEQ ID NO:18) and CDR3 (SEQ ID NO: 19) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 5B shows the nucleotide sequence (SEQ ID NO: 143) and amino acidsequence (SEQ ID NO: 20) of the light chain variable region of the 4C3-1human monoclonal antibody. The CDR1 (SEQ ID NO: 21), CDR2 (SEQ ID NO:22) and CDR3 (SEQ ID NO: 23) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 6A shows the nucleotide sequence (SEQ ID NO: 142) and amino acidsequence (SEQ ID NO: 16) of the heavy chain variable region of the 4C3-2human monoclonal antibody. The CDR1 (SEQ ID NO: 17), CDR2 (SEQ ID NO:18) and CDR3 (SEQ ID NO: 19) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 6B shows the nucleotide sequence (SEQ ID NO: 144) and amino acidsequence (SEQ ID NO: 24) of the light chain variable region of the 4C3-2human monoclonal antibody. The CDR1 (SEQ ID NO: 25), CDR2 (SEQ ID NO:26) and CDR3 (SEQ ID NO: 27) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 7A shows the nucleotide sequence (SEQ ID NO: 142) and amino acidsequence (SEQ ID NO: 16) of the heavy chain variable region of the 4C3-3human monoclonal antibody. The CDR1 (SEQ ID NO: 17), CDR2 (SEQ ID NO:18) and CDR3 (SEQ ID NO: 19) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 7B shows the nucleotide sequence (SEQ ID NO: 145) and amino acidsequence (SEQ ID NO: 28) of the light chain variable region of the 4C3-3human monoclonal antibody. The CDR1 (SEQ ID NO: 29), CDR2 (SEQ ID NO:30) and CDR3 (SEQ ID NO: 31) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 8A shows the nucleotide sequence (SEQ ID NO: 146) and amino acidsequence (SEQ ID NO: 32) of the heavy chain variable region of the 4D4-1human monoclonal antibody. The CDR1 (SEQ ID NO: 33), CDR2 (SEQ ID NO:34) and CDR3 (SEQ ID NO: 35) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 8B shows the nucleotide sequence (SEQ ID NO: 147) and amino acidsequence (SEQ ID NO: 36) of the light chain variable region of the 4D4-1human monoclonal antibody. The CDR1 (SEQ ID NO: 37), CDR2 (SEQ ID NO:38) and CDR3 (SEQ ID NO: 39) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 9A shows the nucleotide sequence (SEQ ID NO: 148) and amino acidsequence (SEQ ID NO: 40) of the heavy chain variable region of the10D2-1 human monoclonal antibody. The CDR1 (SEQ ID NO: 41), CDR2 (SEQ IDNO: 42) and CDR3 (SEQ ID NO: 43) regions are delineated and the V, D andJ germline derivations are indicated.

FIG. 9B shows the nucleotide sequence (SEQ ID NO: 149) and amino acidsequence (SEQ ID NO: 44) of the light chain variable region of the10D2-1 human monoclonal antibody. The CDR1 (SEQ ID NO: 45), CDR2 (SEQ IDNO: 46) and CDR3 (SEQ ID NO: 47) regions are delineated and the V, D andJ germline derivations are indicated.

FIG. 10A shows the nucleotide sequence (SEQ ID NO: 148) and amino acidsequence (SEQ ID NO: 40) of the heavy chain variable region of the10D2-2 human monoclonal antibody. The CDR1 (SEQ ID NO: 41), CDR2 (SEQ IDNO: 42) and CDR3 (SEQ ID NO: 43) regions are delineated and the V, D andJ germline derivations are indicated.

FIG. 10B shows the nucleotide sequence (SEQ ID NO: 150) and amino acidsequence (SEQ ID NO: 48) of the light chain variable region of the10D2-2 human monoclonal antibody. The CDR1 (SEQ ID NO: 49), CDR2 (SEQ IDNO: 50) and CDR3 (SEQ ID NO: 51) regions are delineated and the V, D andJ germline derivations are indicated.

FIG. 11A shows the nucleotide sequence (SEQ ID NO: 151) and amino acidsequence (SEQ ID NO: 52) of the heavy chain variable region of the11A6-1 human monoclonal antibody. The CDR1 (SEQ ID NO: 53), CDR2 (SEQ IDNO: 54) and CDR3 (SEQ ID NO: 55) regions are delineated and the V, D andJ germline derivations are indicated.

FIG. 11B shows the nucleotide sequence (SEQ ID NO: 152) and amino acidsequence (SEQ ID NO: 56) of the light chain variable region of the11A6-1 human monoclonal antibody. The CDR1 (SEQ ID NO: 57), CDR2 (SEQ IDNO: 58) and CDR3 (SEQ ID NO: 59) regions are delineated and the V, D andJ germline derivations are indicated.

FIG. 12A shows the nucleotide sequence (SEQ ID NO: 153) and amino acidsequence (SEQ ID NO: 60) of the heavy chain variable region of the24H2-1 human monoclonal antibody. The CDR1 (SEQ ID NO: 61), CDR2 (SEQ IDNO: 62) and CDR3 (SEQ ID NO: 63) regions are delineated and the V, D andJ germline derivations are indicated.

FIG. 12B shows the nucleotide sequence (SEQ ID NO: 154) and amino acidsequence (SEQ ID NO: 64) of the light chain variable region of the24H2-1 human monoclonal antibody. The CDR1 (SEQ ID NO: 65), CDR2 (SEQ IDNO: 66) and CDR3 (SEQ ID NO: 67) regions are delineated and the V, D andJ germline derivations are indicated.

FIG. 13A shows the nucleotide sequence (SEQ ID NO: 155) and amino acidsequence (SEQ ID NO: 68) of the heavy chain variable region of the 5F8-1human monoclonal antibody. The CDR1 (SEQ ID NO: 69), CDR2 (SEQ ID NO:70) and CDR3 (SEQ ID NO: 71) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 13B shows the nucleotide sequence (SEQ ID NO: 156) and amino acidsequence (SEQ ID NO: 72) of the light chain variable region of the 5F8-1human monoclonal antibody. The CDR1 (SEQ ID NO: 73), CDR2 (SEQ ID NO:74) and CDR3 (SEQ ID NO: 75) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 14A shows the nucleotide sequence (SEQ ID NO: 155) and amino acidsequence (SEQ ID NO: 68) of the heavy chain variable region of the 5F8-2human monoclonal antibody. The CDR1 (SEQ ID NO: 69), CDR2 (SEQ ID NO:70) and CDR3 (SEQ ID NO: 71) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 14B shows the nucleotide sequence (SEQ ID NO: 157) and amino acidsequence (SEQ ID NO: 76) of the light chain variable region of the 5F8-2human monoclonal antibody. The CDR1 (SEQ ID NO: 77), CDR2 (SEQ ID NO:78) and CDR3 (SEQ ID NO: 79) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 15A shows the nucleotide sequence (SEQ ID NO: 155) and amino acidsequence (SEQ ID NO: 68) of the heavy chain variable region of the 5F8-3human monoclonal antibody. The CDR1 (SEQ ID NO: 69), CDR2 (SEQ ID NO:70) and CDR3 (SEQ ID NO: 71) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 15B shows the nucleotide sequence (SEQ ID NO: 242) and amino acidsequence (SEQ ID NO: 238) of the light chain variable region of the5F8-3 human monoclonal antibody. The CDR1 (SEQ ID NO: 239), CDR2 (SEQ IDNO: 240) and CDR3 (SEQ ID NO: 241) regions are delineated and the V, Dand J germline derivations are indicated.

FIG. 16A shows the nucleotide sequence (SEQ ID NO: 158) and amino acidsequence (SEQ ID NO: 80) of the heavy chain variable region of the6E11-1 human monoclonal antibody. The CDR1 (SEQ ID NO: 81), CDR2 (SEQ IDNO: 82) and CDR3 (SEQ ID NO: 83) regions are delineated and the V, D andJ germline derivations are indicated.

FIG. 16B shows the nucleotide sequence (SEQ ID NO: 159) and amino acidsequence (SEQ ID NO: 84) of the light chain variable region of the6E11-1 human monoclonal antibody. The CDR1 (SEQ ID NO: 85), CDR2 (SEQ IDNO: 86) and CDR3 (SEQ ID NO: 87) regions are delineated and the V, D andJ germline derivations are indicated.

FIG. 17A shows the nucleotide sequence (SEQ ID NO: 160) and amino acidsequence (SEQ ID NO: 88) of the heavy chain variable region of the7A11-1 human monoclonal antibody. The CDR1 (SEQ ID NO: 89), CDR2 (SEQ IDNO: 90) and CDR3 (SEQ ID NO: 91) regions are delineated and the V, D andJ germline derivations are indicated.

FIG. 17B shows the nucleotide sequence (SEQ ID NO: 161) and amino acidsequence (SEQ ID NO: 92) of the light chain variable region of the7A11-1 human monoclonal antibody. The CDR1 (SEQ ID NO: 93), CDR2 (SEQ IDNO: 94) and CDR3 (SEQ ID NO: 95) regions are delineated and the V, D andJ germline derivations are indicated.

FIG. 18 shows the amino acid sequence (SEQ ID NO: 189) of the heavychain of anti-CD73 antibody CD73.4-IgG2CS-IgG1.1f, and its variableregion, CDRs 1, 2 and 3, CH1, Hinge, CH2 and CH3 domains.

FIG. 19 shows SPR sensorgram data for the binding of 600, 200, 66.7,22.2, 7.4, and 2.5 nM human-CD73-his (thick lines) or cyno-CD73-his(thin lines) to CD73.4-IgG2-C219S-IgG1.1f captured on an immobilizedprotein A surface at 25° C.

FIGS. 20A1 and 20A2 show the binding of the 11F11, CD73.4 and CD73.10antibodies with the indicated heavy chain constant regions to human CD73positive Calu6 cells (human pulmonary adenocarcinoma cell line).

FIGS. 20B1 and 20B2 show the binding of the 11F11, CD73.4 and CD73.10antibodies with the indicated heavy chain constant regions to human CD73negative DMS114 cells (small lung cell carcinoma cell line).

FIGS. 20C1 and 20C2 show the binding of the 11F11, CD73.4 and CD73.10antibodies with the indicated heavy chain constant regions to cyno CD73positive CHO cells.

FIGS. 20D1 and 20D2 show the binding of the 11F11, CD73.4 and CD73.10antibodies with the indicated heavy chain constant regions to cyno CD73negative CHO-K1 cells.

FIG. 20E shows the binding of the indicated antibodies to T cells fromdonors D1 and D2.

FIG. 20F shows the binding of the indicated antibodies to T cells fromdonors D1 and D2.

FIGS. 21A1 and 21A2 show the inhibition of bead bound human CD73enzymatic activity by the anti-CD73 antibodies 11F11, CD73.4 and CD73.10with the indicated heavy chain constant regions. All antibodiesinhibited human CD73 enzymatic activity.

FIGS. 21B1 and 21B2 show the inhibition of bead bound cyno CD73enzymatic activity by the anti-CD73 antibodies 11F11, CD73.4 and CD73.10with the indicated heavy chain constant regions. All antibodiesinhibited cyno CD73 enzymatic activity.

FIGS. 22A1 and 22A2 show CD73 enzymatic inhibition in human CD73positive Calu6 cells by the 11F11, CD73.4 and CD73.10 antibodies withthe indicated heavy chain constant regions. All antibodies inhibitedCD73 enzymatic activity in these cells.

FIGS. 22B1 and 22B2 show CD73 enzymatic inhibition in human CD73negative DMS-114 cells by the 11F11, CD73.4 and CD73.10 antibodies withthe indicated heavy chain constant regions.

FIG. 22C shows EC50 and Ymax values of inhibition of endogenous CD73activity by 11F11 and 11F11 F(ab′)₂ fragments, as determined in cAMPassay using Calu-6 and HEK/A2R cells. FIG. 22C also shows the EC50 andYmax values of 11F11 and 11F11 F(ab′)2 fragments in a Calu-6internalization assay. The Figure shows that an 11F11 Fab fragment isinactive in these two assays.

FIG. 22D shows a time course of adenosine production from Calu6 cellstreated with the 11F11 or 4C3 antibody, as measured by LC/MS/MS,indicating that CD73 enzymatic inhibition by the 11F11 antibody occursfaster than that by the 4C3 antibody.

FIG. 23A shows the kinetics of antibody mediated internalization of CD73by the following antibodies: 11F11, 4C3, 6D11, CD73.3-IgG1.1f with the4C3Vk1 light chain (“3-Vh-hHC-IgG1.1f/4C3Vk1”), CD73.4-IgG2CS with the11F11 Vk2 light chain (“4-Vh-hHC-IgG2-C219S/11F11-Vk2”), CD73.10-IgG2CS(“CD73.10-Vh-hHC-IgG2-C219S”), CD73.10-IgG2CS-IgG1.1f(“CD73.10-Vh-hHC-IgG2-C219S-IgG1.1f”), and CD73.10-IgG1.1f(“CD73.10-Vh-hHC-IgG1.1f”) antibodies in H2228 cells. The 11F11 (whichis of an IgG2 isotype), CD73.4-IgG2CS, CD73.10-IgG2CS andCD73.10-IgG2CS-IgG1.1f antibodies are internalized faster and to ahigher degree than the other tested antibodies, which are of an IgG1isotype.

FIG. 23B shows the kinetics of antibody mediated CD73 internalization ofthe same antibodies as those shown in FIG. 23A in HCC15 cells (non-smallcell lung carcinoma cell line), showing similar results to thoseobtained in H2228 cells (non-small cell lung carcinoma cell line).

FIG. 23C shows the kinetics of antibody mediated CD73 internalization ofthe same antibodies as those shown in FIGS. 23A and 23B, as well asCD73.11-IgG2CS (“11-Vh-hVC-IgG2-C219S”), in Calu6 cells, showing similarresults to those obtained in H2228 and HCC15 cells.

FIG. 23D shows the kinetics of antibody mediated CD73 internalization ofthe same antibodies as those shown in FIG. 23C in NCI-2030 cells(non-small cell lung carcinoma cell line), showing similar results tothose obtained in H2228, HCC15, and Calu6 cells.

FIG. 23E shows the kinetics of antibody mediated CD73 internalization ofthe indicated antibodies in Calu6 cells, as measured by flow cytometry.

FIG. 23F shows the kinetics of antibody mediated CD73 internalization ofthe indicated antibodies in NCI-H292 cells (mucoepidermoid pulmonarycarcinoma cell line), as measured by flow cytometry, but where theantibodies were not washed out after the first incubation of the cellswith the antibodies.

FIG. 23G shows the percentage of CD73 internalized in Calu6 cellstreated with the indicated antibodies, showing antibody mediated CD73internalization of the indicated antibodies in Calu6 cells over time.

FIG. 23H shows the percentage of CD73 internalized in NCI-H292 cellstreated with the indicated antibodies over time, showing antibodymediated CD73 internalization of the indicated antibodies in NCI-H292cells over time.

FIG. 23I shows the percentage of CD73 internalized in SNU-C1 cells(colon carcinoma cell line) treated with the indicated antibodies overtime, showing antibody mediated CD73 internalization of the indicatedantibodies in SNU-C1 cells over time.

FIG. 23J shows the percentage of CD73 internalized in NCI-H1437 cells(non-small cell lung carcinoma cell line) treated with the indicatedantibodies over time, showing antibody mediated CD73 internalization ofthe indicated antibodies in NCI-H1437 cells over time.

FIG. 23K shows the percentage of CD73 internalized in Calu6 cellstreated with the indicated antibodies over time, showing antibodymediated CD73 internalization of the indicated antibodies in Calu6 cellsover time.

FIG. 23L shows the percentage of CD73 internalized in NCI-H292 cellstreated with the indicated antibodies over time, showing antibodymediated CD73 internalization of the indicated antibodies in Calu6 cellsover time.

FIG. 23M shows the level of CD73 on the surface of Calu6 cells treatedwith 5 μg/ml of the indicated antibodies for 0, 5, 15 or 30 minutes.

FIG. 24A shows xenograft tumor sections from animals harvested 4 daysafter treatment of the animals with a control antibody and stained forCD73 enzymatic activity. The sections show a dense brown color,indicating CD73 enzymatic activity.

FIG. 24B shows xenograft tumor sections from animals harvested 1 dayafter treatment of the animals with the 11F11 antibody and stained forCD73 enzymatic activity. The sections show significantly less browncolor relative to the control tumor sections shown in FIG. 24A,indicating in vivo inhibition of CD73 enzymatic activity byCD73.10-IgG2CS-IgG1.1f as early as 1 day after the start of thetreatment.

FIG. 24C shows xenograft tumor sections from animals harvested 2 daysafter treatment of the animals with CD73.10-IgG2CS-IgG1.1f and stainedfor CD73 enzymatic activity. The sections show significantly less browncolor relative to the control tumor sections shown in FIG. 24A andrelative to the tumor sections after 1 day of treatment of the animalswith CD73.10-IgG2CS-IgG1.1f, indicating in vivo inhibition of CD73enzymatic activity by CD73.10-IgG2CS-IgG1.1f at least 2 days after thestart of the treatment.

FIG. 24D shows xenograft tumor sections from animals harvested 3 daysafter treatment of the animals with CD73.10-IgG2CS-IgG1.1f and stainedfor CD73 enzymatic activity. The sections show significantly less browncolor relative to the control tumor sections shown in FIG. 24A,indicating in vivo inhibition of CD73 enzymatic activity byCD73.10-IgG2CS-IgG1.1f at least 3 days after the start of the treatment.

FIG. 24E shows xenograft tumor sections from animals harvested 7 daysafter treatment of the animals with CD73.10-IgG2CS-IgG1.1f and stainedfor CD73 enzymatic activity. The sections show significantly less browncolor relative to the control tumor sections shown in FIG. 24A,indicating in vivo inhibition of CD73 enzymatic activity byCD73.10-IgG2CS-IgG1.1f at least 7 days after the start of the treatment.

FIG. 24F shows a time course of the enzymatic activity of human CD73 inSNUC1 tumors in xenograft mice treated with a control (non CD73)antibody or with 1 mg/kg, 3 mg/kg or 10 mg/kg CD73.4-IgG2CS-IgG1.1f,showing that the anti-CD73 antibody efficiently reduces CD73 enzymaticactivity in the tumors of the xenograft mice.

FIG. 25A shows levels of mouse CD73 enzymatic activity in control tumorsections from Balb/c mice bearing syngeneic 4T1 tumors and control mIgG.

FIG. 25B shows tumor sections (4T1 days 1-7) of Balb/c mice bearingsyngeneic 4T1 tumors subcutaneously treated with anti-mouse CD73antibody TY23, showing that TY23 inhibits CD73 enzymatic inhibition invivo.

FIG. 26A shows the level of cross-blocking of 4C3 by the anti-CD73antibodies 4C3, 7A11, 6E11, 5F8, 4C3, 11F11 and 11A6 as determined byflow cytometry.

FIG. 26B shows the level of cross-blocking of 11F11 by the anti-CD73antibodies 4C3, 7A11, 6E11, 5F8, 4C3, 11F11 and 11A6 as determined byflow cytometry.

FIG. 27A shows the amino acid sequence (SEQ ID NO: 283) of human CD73and the regions of interaction with CD73.4-IgG2CS-IgG1.1f, which arerepresented in a darker grey. The stronger the interaction, the darkerthe grey.

FIG. 27B shows a model of the interaction between a dimeric human CD73protein and CD73.4-IgG2CS-IgG1.1f.

FIG. 28A shows a crystallographic model of the interaction between humanCD73 and 11F11Fab′ fragment.

FIG. 28B shows a model of a composite structure of two human CD73complexes with 11F11.

FIG. 28C shows a model of the interaction between human CD73 and 11F11antibody.

FIG. 28D shows a model of the interaction between 11F11 and human CD73.

FIG. 29A shows SEC-MALS data for human CD73 and antibody complexes.“CD73.4-hybrid” refers to CD73.4-IgG2CS-IgG1.1f.

FIG. 29B shows DLS data for human CD73 and antibody complexes.

FIG. 30A shows SEC chromatogram data for complexes of hCD73-his with theCD73.4 antibody containing different constant regions, showing theeffect of an IgG2 hinge and CH1 domain on the size of antibody/antigencomplexes.

FIG. 30B shows DLS data for complexes of hCD73-his with the CD73.4antibody containing different constant regions, showing the effect of anIgG2 hinge and CH1 domain on the size of antibody/antigen complexes.

FIG. 30C shows MALS data for complexes of hCD73-his with the CD73.4antibody containing different constant regions, showing the effect of anIgG2 hinge and CH1 domain on the size of antibody/antigen complexes.

FIG. 30D shows a schematic model of the hCD73-his/mAb complexes derivedfrom the MALS-determined masses in FIG. 30C.

FIG. 30E shows that higher order complexes are impacted by the CH1region. The histograms show the % area under peaks 1 and 2, shown in thegraph, for each construct.

FIG. 31 shows the percentage of antibody mediated CD73 internalizationat 1, 4 or 21 hours after the addition of each of the shown antibodies.The bars for each antibody are shown in the order of 21 hours (on theleft), 4 hours (middle) and 1 hour (right).

FIG. 32A shows an overlay of SEC chromatogram data for 1:1 molarcomplexes of hCD73-his with 16 different CD73.4 antibodies containingdifferent constant region sequences.

FIG. 32B shows an expansion of the chromatogram data from 11-19.5 min ofthe chromatogram of FIG. 32A, with 4 distinct elution species indicated.

FIG. 32C shows the percentage of the UV chromatogram signal area forpeak 2 of FIG. 32B, plotted for the 16 different antibody/CD73-hiscomplexes. Data is sorted from left to right in order of increasing peakarea.

FIG. 33 shows antibody binding to anti-his Fab captured FcγR-hisproteins. Binding responses are plotted as a percentage of thetheoretical Rmax assuming a 1:1 mAb:FcγR binding stoichiometry. The barsfor each antibody are shown in the order provided by the color legendsat the bottom of the slide.

FIG. 34 shows antibody binding to anti-his Fab captured FcgR-hisproteins. Binding responses are plotted as a percentage of thetheoretical Rmax assuming a 1:1 mAb:FcγR binding stoichiometry. The barsfor each antibody are shown in the order provided by the color legendsat the bottom of the slide.

FIG. 35 shows an alignment of the VH and VL sequences of variousanti-CD73 antibodies. VH and VL CDR1, CDR2 and CDR3 sequences arebolded.

DETAILED DESCRIPTION

Described herein are isolated antibodies, particularly monoclonalantibodies, e.g., human monocloncal antibodies, which specifically bindto CD73 and thereby reduce CD73 activity (“antagonist anti-CD73antibodies”). In certain embodiments, the antibodies described hereinare derived from particular heavy and light chain germline sequencesand/or comprise particular structural features such as CDR regionscomprising particular amino acid sequences. Provided herein are isolatedantibodies, methods of making such antibodies, immunoconjugates andbispecific molecules comprising such antibodies, and pharmaceuticalcompositions formulated to contain the antibodies. Also provided hereinare methods of using the antibodies for reducing tumor growth, alone orin combination with other therapeutic agents (e.g., antibodies) and/orcancer therapies. Accordingly, the anti-CD73 antibodies described hereinmay be used in a treatment in a wide variety of therapeuticapplications, including, for example, inhibition of tumor growth,inhibition of metastasis, and enhancement of an immune response againsta tumor.

Definitions

In order that the present description may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The term “Cluster of Differentiation 73” or “CD73” as used herein refersto an enzyme (nucleotidase) capable of converting extracellularnucleoside 5′ monophosphates to nucleosides, namely adenosinemonophosphate (AMP) to adenosine. CD73 is usually found as a dimeranchored to the cell membrane through a glycosylphosphatidylinositol(GPI) linkage, has ecto-enzyme activity and plays a role in signaltransduction. The primary function of CD73 is its conversion ofextracellular nucleotides (e.g., 5′-AMP) to adenosine, a highlyimmunosuppressive molecule. Thus, ecto-5′-nucleotidase catalyzes thedephosphorylation of purine and pyrimidine ribo- and deoxyribonulceosidemonophosphates to the corresponding nucleoside. Although CD73 has broadsubstrate specificity, it prefers purine ribonucleosides.

CD73 is also referred to as ecto-5′nuclease (ecto-5′NT, EC 3.1.3.5). Theterm “CD73” includes any variants or isoforms of CD73 which arenaturally expressed by cells. Accordingly, antibodies described hereinmay cross-react with CD73 from species other than human (e.g.,cynomolgus CD73). Alternatively, the antibodies may be specific forhuman CD73 and may not exhibit any cross-reactivity with other species.CD73 or any variants and isoforms thereof, may either be isolated fromcells or tissues which naturally express them or be recombinantlyproduced using well-known techniques in the art and/or those describedherein.

Two isoforms of human CD73 have been identified, both of which share thesame N-terminal and C-terminal portions. Isoform 1 (Accession No. NP002517.1; SEQ ID NO: 1) represents the longest protein, consisting of574 amino acids and 9 exons. Isoform 2 (Accession No. NP 001191742.1;SEQ ID NO: 2) encodes a shorter protein, consisting of 524 amino acids,lacking amino acids 404-453. Isoform 2 lacks an alternate in-frame exonresulting in a transcript with only 8 exons, but with the same N- andC-terminal sequences.

The cynomolgus (cyno) CD73 protein sequence is provided as SEQ ID NO: 3.The terms cynomolgus and cyno both refer to the Macaca fascicularisspecies and are use interchangably throughout the specification.

The term “antibody” as used herein may include whole antibodies and anyantigen binding fragments (i.e., “antigen-binding portions”) or singlechains thereof. An “antibody” refers, in one embodiment, to aglycoprotein comprising at least two heavy (H) chains and two light (L)chains inter-connected by disulfide bonds, or an antigen binding portionthereof. Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as V_(H)) and a heavy chain constant region. Incertain naturally occurring IgG, IgD and IgA antibodies, the heavy chainconstant region is comprised of three domains, CH1, CH2 and CH3. Incertain naturally occurring antibodies, each light chain is comprised ofa light chain variable region (abbreviated herein as V_(L)) and a lightchain constant region. The light chain constant region is comprised ofone domain, CL. The V_(H) and V_(L) regions can be further subdividedinto regions of hypervariability, termed complementarity determiningregions (CDR), interspersed with regions that are more conserved, termedframework regions (FR). Each V_(H) and V_(L) is composed of three CDRsand four FRs, arranged from amino-terminus to carboxy-terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variableregions of the heavy and light chains contain a binding domain thatinteracts with an antigen. The constant regions of the antibodies maymediate the binding of the immunoglobulin to host tissues or factors,including various cells of the immune system (e.g., effector cells) andthe first component (Clq) of the classical complement system.

The heavy chain of an antibody may or may not contain a terminal lysine(K), or a terminal glycine and lysine (GK). Thus, any of the heavy chainsequences and heavy chain constant region sequences provided herein canend in either GK or G, or lack K or GK, regardless of what the lastamino acid of the sequence provides. This is because the terminal lysineand sometimes glycine and lysine are cleaved during expression of theantibody.

Antibodies typically bind specifically to their cognate antigen withhigh affinity, reflected by a dissociation constant (K_(D)) of 10 to10⁻¹¹ M or less. Any K_(D) greater than about 10⁻⁶ M is generallyconsidered to indicate nonspecific binding. As used herein, an antibodythat “binds specifically” to an antigen refers to an antibody that bindsto the antigen and substantially identical antigens with high affinity,which means having a K_(D) of 10⁻⁷ M or less, preferably 10⁻⁸ M or less,even more preferably 5×10⁻⁹ M or less, and most preferably between 10⁻⁸Mand 10⁻¹⁰ M or less, but does not bind with high affinity to unrelatedantigens. An antigen is “substantially identical” to a given antigen ifit exhibits a high degree of sequence identity to the given antigen, forexample, if it exhibits at least 80%, at least 90%, at least 95%, atleast 97%, or at least 99% or greater sequence identity to the sequenceof the given antigen. By way of example, an antibody that bindsspecifically to human CD73 may also cross-react with CD73 from certainnon-human primate species (e.g., cynomolgus monkey), but may notcross-react with CD73 from other species, or with an antigen other thanCD73.

An immunoglobulin may be from any of the commonly known isotypes,including but not limited to IgA, secretory IgA, IgG and IgM. The IgGisotype is divided in subclasses in certain species: IgG1, IgG2, IgG3and IgG4 in humans, and IgG1, IgG2a, IgG2b and IgG3 in mice. In certainembodiments, the anti-CD73 antibodies described herein are of the humanIgG1 or IgG2 subtype. Immunoglobulins, e.g., human IgG1, exist inseveral allotypes, which differ from each other in at most a few aminoacids. “Antibody” may include, by way of example, both naturallyoccurring and non-naturally occurring antibodies; monoclonal andpolyclonal antibodies; chimeric and humanized antibodies; human andnonhuman antibodies; wholly synthetic antibodies; and single chainantibodies.

The term “antigen-binding portion” of an antibody, as used herein,refers to one or more fragments of an antibody that retain the abilityto specifically bind to an antigen (e.g., human CD73). It has been shownthat the antigen-binding function of an antibody can be performed byfragments of a full-length antibody. Examples of binding fragmentsencompassed within the term “antigen-binding portion” of an antibody,e.g., an anti-CD73 antibody described herein, include (i) a Fabfragment, a monovalent fragment consisting of the V_(L), V_(H), CL andCH1 domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising twoFab fragments linked by a disulfide bridge at the hinge region; (iii) aFd fragment consisting of the V_(H) and CH1 domains; (iv) a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546),which consists of a V_(H) domain; and (vi) an isolated complementaritydetermining region (CDR) or (vii) a combination of two or more isolatedCDRs which may optionally be joined by a synthetic linker. Furthermore,although the two domains of the Fv fragment, V_(L) and V_(H), are codedfor by separate genes, they can be joined, using recombinant methods, bya synthetic linker that enables them to be made as a single proteinchain in which the V_(L) and V_(H) regions pair to form monovalentmolecules known as single chain Fv (scFv); see e.g., Bird et al. (1988)Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.These and other potential constructs are described at Chan & Carter(2010) Nat. Rev. Immunol. 10:301. These antibody fragments are obtainedusing conventional techniques known to those with skill in the art, andthe fragments are screened for utility in the same manner as are intactantibodies. Antigen-binding portions can be produced by recombinant DNAtechniques, or by enzymatic or chemical cleavage of intactimmunoglobulins.

A “bispecific” or “bifunctional antibody” is an artificial hybridantibody having two different heavy/light chain pairs, giving rise totwo antigen binding sites with specificity for different antigens.Bispecific antibodies can be produced by a variety of methods includingfusion of hybridomas or linking of Fab′ fragments. See, e.g.,Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelnyet al., J. Immunol. 148, 1547-1553 (1992).

The term “monoclonal antibody,” as used herein, refers to an antibodythat displays a single binding specificity and affinity for a particularepitope or a composition of antibodies in which all antibodies display asingle binding specificity and affinity for a particular epitope.Typically such monoclonal antibodies will be derived from a single cellor nucleic acid encoding the antibody, and will be propagated withoutintentionally introducing any sequence alterations. Accordingly, theterm “human monoclonal antibody” refers to a monoclonal antibody thathas variable and optional constant regions derived from human germlineimmunoglobulin sequences. In one embodiment, human monoclonal antibodiesare produced by a hybridoma, for example, obtained by fusing a B cellobtained from a transgenic or transchromosomal non-human animal (e.g., atransgenic mouse having a genome comprising a human heavy chaintransgene and a light chain transgene), to an immortalized cell.

The term “recombinant human antibody,” as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as (a) antibodies isolated from an animal (e.g.,a mouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom, (b) antibodies isolated from ahost cell transformed to express the antibody, e.g., from atransfectoma, (c) antibodies isolated from a recombinant, combinatorialhuman antibody library, and (d) antibodies prepared, expressed, createdor isolated by any other means that involve splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies comprise variable and constant regions that utilizeparticular human germline immunoglobulin sequences and are encoded bythe germline genes, but include subsequent rearrangements and mutationsthat occur, for example, during antibody maturation. As known in the art(see, e.g., Lonberg (2005) Nature Biotech. 23(9):1117-1125), thevariable region contains the antigen binding domain, which is encoded byvarious genes that rearrange to form an antibody specific for a foreignantigen. In addition to rearrangement, the variable region can befurther modified by multiple single amino acid changes (referred to assomatic mutation or hypermutation) to increase the affinity of theantibody to the foreign antigen. The constant region will change infurther response to an antigen (i.e isotype switch). Therefore, therearranged and somatically mutated nucleic acid sequences that encodethe light chain and heavy chain immunoglobulin polypeptides in responseto an antigen may not be identical to the original germline sequences,but instead will be substantially identical or similar (i.e., have atleast 80% identity).

A “human” antibody (HuMAb) refers to an antibody having variable regionsin which both the framework and CDR regions are derived from humangermline immunoglobulin sequences. Furthermore, if the antibody containsa constant region, the constant region also is derived from humangermline immunoglobulin sequences. The antibodies described herein mayinclude amino acid residues not encoded by human germline immunoglobulinsequences (e.g., mutations introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo). However, the term“human antibody”, as used herein, is not intended to include antibodiesin which CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences. The terms “human” antibodies and “fully human” antibodies andare used synonymously.

A “humanized” antibody refers to an antibody in which some, most or allof the amino acids outside the CDR domains of a non-human antibody arereplaced with corresponding amino acids derived from humanimmunoglobulins. In one embodiment of a humanized form of an antibody,some, most or all of the amino acids outside the CDR domains have beenreplaced with amino acids from human immunoglobulins, whereas some, mostor all amino acids within one or more CDR regions are unchanged. Smalladditions, deletions, insertions, substitutions or modifications ofamino acids are permissible as long as they do not abrogate the abilityof the antibody to bind to a particular antigen. A “humanized” antibodyretains an antigenic specificity similar to that of the originalantibody.

A “chimeric antibody” refers to an antibody in which the variableregions are derived from one species and the constant regions arederived from another species, such as an antibody in which the variableregions are derived from a mouse antibody and the constant regions arederived from a human antibody.

A “modified heavy chain constant region” refers to a heavy chainconstant region comprising the constant domains CH1, hinge, CH2, andCH3, wherein one or more of the constant domains are from a differentisotype (e.g. IgG1, IgG2, IgG3, IgG4). In certain embodiments, themodified constant region includes a human IgG2 CH1 domain and a humanIgG2 hinge fused to a human IgG1 CH2 domain and a human IgG1 CH3 domain.In certain embodiments, such modified constant regions also includeamino acid modifications within one or more of the domains relative tothe wildtype amino acid sequence.

When referring herein to an antibody as “CD73.3” or “CD73.4” withoutindicating the identity of the constant region, unless otherwiseindicated, refers to antibodies having the variable regions of CD73.3 orCD73.4, respectively, with any constant region described herein.

As used herein, “isotype” refers to the antibody class (e.g., IgG1,IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibody) that isencoded by the heavy chain constant region genes.

“Allotype” refers to naturally occurring variants within a specificisotype group, which variants differ in a few amino acids (see, e.g.,Jefferis et al. (2009) mAbs 1:1). Antibodies described herein may be ofany allotype.

Unless specified otherwise herein, all amino acid numbers are accordingto the EU index of the Kabat system (Kabat, E. A., et al. (1991)Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242).

The phrases “an antibody recognizing an antigen” and “an antibodyspecific for an antigen” are used interchangeably herein with the term“an antibody which binds specifically to an antigen.”

An “isolated antibody,” as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds to CD73 is substantially free of antibodies that specifically bindantigens other than CD73). An isolated antibody that specifically bindsto an epitope of CD73 may, however, have cross-reactivity to other CD73proteins from different species.

As used herein, an antibody that “inhibits CD73” refers to an antibodythat inhibits a biological and/or enzymatic function of CD73. Thesefunctions include, for example, the ability of an antibody to inhibitCD73 enzymatic activity, e.g., CD73-regulated production of adenosine orreduction of cAMP production.

As used herein, an antibody that “internalizes” refers to an antibodythat crosses the cell membrane upon binding to a cell-surface antigen.Internalization includes antibody mediated receptor, e.g., CD73,internalization. In some embodiments, the antibody “internalizes” intocells expressing CD73 at a rate of T_(1/2) equal to about 10 min orless.

An “effector function” refers to the interaction of an antibody Fcregion with an Fc receptor or ligand, or a biochemical event thatresults therefrom. Exemplary “effector functions” include Clq binding,complement dependent cytotoxicity (CDC), Fc receptor binding,FcγR-mediated effector functions such as ADCC and antibody dependentcell-mediated phagocytosis (ADCP), and downregulation of a cell surfacereceptor (e.g., the B cell receptor; BCR). Such effector functionsgenerally require the Fc region to be combined with a binding domain(e.g., an antibody variable domain).

An “Fc receptor” or “FcR” is a receptor that binds to the Fc region ofan immunoglobulin. FcRs that bind to an IgG antibody comprise receptorsof the FcγR family, including allelic variants and alternatively splicedforms of these receptors. The FcγR family consists of three activating(FcγRI, FcγRIII, and FcγRIV in mice; FcγRIA, FcγRIIA, and FcγRIIIA inhumans) and one inhibitory (FcγRIIB) receptor. Various properties ofhuman FcγRs are summarized in Table 1. The majority of innate effectorcell types coexpress one or more activating FcγR and the inhibitoryFcγRIIB, whereas natural killer (NK) cells selectively express oneactivating Fc receptor (FcγRIII in mice and FcγRIIIA in humans) but notthe inhibitory FcγRIIB in mice and humans. Human IgG1 binds to mosthuman Fc receptors and is considered equivalent to murine IgG2a withrespect to the types of activating Fc receptors that it binds to.

TABLE 1 Properties of human FcγRs Allelic Affinity for Fcγ variantshuman IgG Isotype preference Cellular distribution FcγRI None High(K_(D) ~10 IgG1 = 3 > 4 >> 2 Monocytes, macrophages, described nM)activated neutrophils, dentritic cells? FcγRIIA H131 Low to mediumIgG1 > 3 > 2 > 4 Neutrophils, monocytes, R131 Low IgG1 > 3 > 4 > 2macrophages, eosinophils, dentritic cells, platelets FcγRIIIA V158Medium IgG1 = 3 >> 4 > 2 NK cells, monocytes, F158 Low IgG1 = 3 >> 4 > 2macrophages, mast cells, eosinophils, dentritic cells? FcγRIIB I232 LowIgG1 = 3 = 4 > 2 B cells, monocytes, T232 Low IgG1 = 3 = 4 > 2macrophages, dentritic cells, mast cells

A “hinge”, “hinge domain” or “hinge region” or “antibody hinge region”refers to the domain of a heavy chain constant region that joins the CH1domain to the CH2 domain and includes the upper, middle, and lowerportions of the hinge (Roux et al. J. Immunol. 1998 161:4083). The hingeprovides varying levels of flexibility between the binding and effectorregions of an antibody and also provides sites for intermoleculardisulfide bonding between the two heavy chain constant regions. As usedherein, a hinge starts at Glu216 and ends at Gly237 for all IgG isotypes(Roux et al., 1998 J Immunol 161:4083). The sequences of wildtype IgG1,IgG2, IgG3 and IgG4 hinges are show in Tables 2 and 31.

TABLE 2 Hinge region amino acids C-termi-  Upper  Lower  nal CH1* HingeHinge (SEQ ID (SEQ ID Middle Hinge (SEQ ID Ig Type NO) NO) (SEQ ID NO)NO) IgG1 VDKRV EPKSCDK CPPCP APELLGG (284) THT (290) (298) (286) IgG2VDKTV ERK CCVECPPCP APPVAG (285) (291) (299) IgG3 VDKRV ELKTPLG CPRCPAPELLGG (17-15- (284) DTTHT (EPKSCDTP (298) 15-15) (287) PPCPRCP)₃ (292)IgG3 VDKRV ELKTPLG CPRCP APELLGG (17-15- (284) DTTHT (EPKSCDTP (298) 15)(287) PPCPRCP)₂ (293) IgG3  VDKRV ELKTPLG CPRCP APELLGG (17-15) (284)DTTHT (EPKSCDTP (298) (287) PPCPRCP)₁ (294) IgG3 VDKRV EPKS CDTPPPCPRCPAPELLGG (15-15- (284) (288) (EPKSCDTPPP (298) 15) CPRCP)₂ (295) IgG3VDKRV EPKS CDTPPPCPRCP APELLGG (15) (284) (288) (296) (298) IgG4 VDKRVESKYGPP CPSCP APEFLGG (284) (289) (297) (298)

The term “hinge” includes wildtype hinges (such as those set forth inTables 2 and 31), as well as variants thereof (e.g.,non-naturally-occurring hinges or modified hinges). For example, theterm “IgG2 hinge” includes wildtype IgG2 hinge, as shown in Table 2, andvariants having 1, 2, 3, 4, 5, 1-3, 1-5, 3-5 and/or at most 5, 4, 3, 2,or 1 mutations, e.g., substitutions, deletions or additions. ExemplaryIgG2 hinge variants include IgG2 hinges in which 1, 2, 3 or all 4cysteines (C219, C220, C226 and C229) are changed to another amino acid.In a specific embodiment, an IgG2 comprises a C219S substitution. AnIgG2 hinge may also comprise a substitution at C220 or substitutions atboth C219 and a C220. An IgG2 hinge may comprise a substitution, whichalone, or together with one or more substitutions in other regions ofthe heavy or light chain will cause the antibody to take form A or B(see, e.g., Allen et al. (2009) Biochemistry 48:3755). In certainembodiments, a hinge is a hybrid hinge that comprises sequences from atleast two isotypes. For example, a hinge may comprise the upper, middleor lower hinge from one isotype and the remainder of the hinge from oneor more other isotypes. For example, a hinge can be an IgG2/IgG1 hinge,and may comprise, e.g., the upper and middle hinges of IgG2 and thelower hinge of IgG1. A hinge may have effector function or be deprivedof effector function. For example, the lower hinge of wildtype IgG1provides effector function. *C-terminal amino acid sequences of the CH1domains.

The term “CH1 domain” refers to the heavy chain constant region linkingthe variable domain to the hinge in a heavy chain constant domain. Asused herein, a CH1 domain starts at A118 and ends at V215. The term “CH1domain” includes wildtype CH1 domains (such as having SEQ ID NO: 98 forIgG1 and SEQ ID NO: 124 for IgG2), as well as variants thereof (e.g.,non-naturally-occurring CH1 domains or modified CH1 domains). Forexample, the term “CH1 domain” includes wildtype CH1 domains andvariants thereof having 1, 2, 3, 4, 5, 1-3, 1-5, 3-5 and/or at most 5,4, 3, 2, or 1 mutations, e.g., substitutions, deletions or additions.Exemplary CH1 domains include CH1 domains with mutations that modify abiological activity of an antibody, such as ADCC, CDC or half-life.Modifications to the CH1 domain that affect a biological activity of anantibody are provided herein. A CH1 domain may comprise the substitutionC131S, which substitution may cause an IgG2 antibody or an antibodycomprising at least a portion of an IgG2 antibody, such as the hingeand/or the hinge and CH1, to adopt the B form, as opposed to the A formof the antibody.

The term “CH2 domain” refers to the heavy chain constant region linkingthe hinge to the CH3 domain in a heavy chain constant domain. As usedherein, a CH2 domain starts at P238 and ends at K340. The term “CH2domain” includes wildtype CH2 domains (such as having SEQ ID NO: 137 forIgG1; Table 35), as well as variants thereof (e.g.,non-naturally-occurring CH2 domains or modified CH2 domains). Forexample, the term “CH2 domain” includes wildtype CH2 domains andvariants thereof having 1, 2, 3, 4, 5, 1-3, 1-5, 3-5 and/or at most 5,4, 3, 2, or 1 mutations, e.g., substitutions, deletions or additions.Exemplary CH2 domains include CH2 domains with mutations that modify abiological activity of an antibody, such as ADCC, CDC or half-life. Incertain embodiments, a CH2 domain comprises the substitutionsA330S/P331S that reduce effector function. Other modifications to theCH2 domain that affect a biological activity of an antibody are providedherein.

The term “CH3 domain” refers to the heavy chain constant region that isC-terminal to the CH2 domain in a heavy chain constant domain. As usedherein, a CH3 domain starts at G341 and ends at K447. The term “CH3domain” includes wildtype CH3 domains (such as having SEQ ID NO: 138 forIgG1; Table 35), as well as variants thereof (e.g.,non-naturally-occurring CH3 domains or modified CH3 domains). Forexample, the term “CH3 domain” includes wildtype CH3 domains andvariants thereof having 1, 2, 3, 4, 5, 1-3, 1-5, 3-5 and/or at most 5,4, 3, 2, or 1 mutations, e.g., substitutions, deletions or additions.Exemplary CH3 domains include CH3 domains with mutations that modify abiological activity of an antibody, such as ADCC, CDC or half-life.Modifications to the CH3 domain that affect a biological activity of anantibody are provided herein.

A “CL domain” refers to the constant domain of a light chain. The term“CL domain” includes wildtype CL domains and variants thereof, e.g.,variants comprising C214S.

A “native sequence Fc region” or “native sequence Fc” comprises an aminoacid sequence that is identical to the amino acid sequence of an Fcregion found in nature. Native sequence human Fc regions include anative sequence human IgG1 Fc region; native sequence human IgG2 Fcregion; native sequence human IgG3 Fc region; and native sequence humanIgG4 Fc region as well as naturally occurring variants thereof. Nativesequence Fc includes the various allotypes of Fcs (see, e.g., Jefferiset al. (2009) mAbs 1:1).

The term “epitope” or “antigenic determinant” refers to a site on anantigen (e.g., CD73) to which an immunoglobulin or antibody specificallybinds. Epitopes within protein antigens can be formed both fromcontiguous amino acids (usually a linear epitope) or noncontiguous aminoacids juxtaposed by tertiary folding of the protein (usually aconformational epitope). Epitopes formed from contiguous amino acids aretypically, but not always, retained on exposure to denaturing solvents,whereas epitopes formed by tertiary folding are typically lost ontreatment with denaturing solvents. An epitope typically includes atleast 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in aunique spatial conformation. Methods for determining what epitopes arebound by a given antibody (i.e., epitope mapping) are well known in theart and include, for example, immunoblotting and immunoprecipitationassays, wherein overlapping or contiguous peptides (e.g., from CD73) aretested for reactivity with a given antibody (e.g., anti-CD73 antibody).Methods of determining spatial conformation of epitopes includetechniques in the art and those described herein, for example, x-raycrystallography, 2-dimensional nuclear magnetic resonance and HDX-MS(see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology,Vol. 66, G. E. Morris, Ed. (1996)).

The term “epitope mapping” refers to the process of identification ofthe molecular determinants on the antigen involved in antibody-antigenrecognition.

The term “binds to the same epitope” with reference to two or moreantibodies means that the antibodies bind to the same segment of aminoacid residues, as determined by a given method. Techniques fordetermining whether antibodies bind to the “same epitope on CD73” withthe antibodies described herein include, for example, epitope mappingmethods, such as, x-ray analyses of crystals of antigen:antibodycomplexes, which provides atomic resolution of the epitope, andhydrogen/deuterium exchange mass spectrometry (HDX-MS). Other methodsthat monitor the binding of the antibody to antigen fragments (e.g.proteolytic fragments) or to mutated variations of the antigen whereloss of binding due to a modification of an amino acid residue withinthe antigen sequence is often considered an indication of an epitopecomponent (e.g. alanine scanning mutagenesis—Cunningham & Wells (1985)Science 244:1081). In addition, computational combinatorial methods forepitope mapping can also be used. These methods rely on the ability ofthe antibody of interest to affinity isolate specific short peptidesfrom combinatorial phage display peptide libraries.

Antibodies that “compete with another antibody for binding to a target”refer to antibodies that inhibit (partially or completely) the bindingof the other antibody to the target. Whether two antibodies compete witheach other for binding to a target, i.e., whether and to what extent oneantibody inhibits the binding of the other antibody to a target, may bedetermined using known competition experiments, e.g., such as thosedescribed in the Examples. In certain embodiments, an antibody competeswith, and inhibits binding of another antibody to a target by at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. The level ofinhibition or competition may be different depending on which antibodyis the “blocking antibody” (i.e., the cold antibody that is incubatedfirst with the target). Competition assays can be conducted asdescribed, for example, in Ed Harlow and David Lane, Cold Spring HarbProtoc; 2006; doi:10.1101/pdb.prot4277 or in Chapter 11 of “UsingAntibodies” by Ed Harlow and David Lane, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., USA 1999. Competing antibodies bind tothe same epitope, an overlapping epitope or to adjacent epitopes (e.g.,as evidenced by steric hindrance).

Other competitive binding assays include: solid phase direct or indirectradioimmunoassay (MA), solid phase direct or indirect enzyme immunoassay(EIA), sandwich competition assay (see Stahli et al., Methods inEnzymology 9:242 (1983)); solid phase direct biotin-avidin EIA (seeKirkland et al., J. Immunol. 137:3614 (1986)); solid phase directlabeled assay, solid phase direct labeled sandwich assay (see Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988));solid phase direct label RIA using 1-125 label (see Morel et al., Mol.Immunol. 25(1):7 (1988)); solid phase direct biotin-avidin EIA (Cheunget al., Virology 176:546 (1990)); and direct labeled RIA. (Moldenhaueret al., Scand. J. Immunol. 32:77 (1990)).

As used herein, the terms “specific binding,” “selective binding,”“selectively binds,” and “specifically binds,” refer to antibody bindingto an epitope on a predetermined antigen but not to other antigens.Typically, the antibody (i) binds with an equilibrium dissociationconstant (K_(D)) of approximately less than 10⁻⁷ M, such asapproximately less than 10⁻⁸ M, 10⁻⁹ M or 10⁻¹⁰ M or even lower whendetermined by, e.g., surface plasmon resonance (SPR) technology in aBIACORE® 2000 surface plasmon resonance instrument using thepredetermined antigen, e.g., recombinant human CD73, as the analyte andthe antibody as the ligand, or Scatchard analysis of binding of theantibody to antigen positive cells, and (ii) binds to the predeterminedantigen with an affinity that is at least two-fold greater than itsaffinity for binding to a non-specific antigen (e.g., BSA, casein) otherthan the predetermined antigen or a closely-related antigen.Accordingly, unless otherwise indicated, an antibody that “specificallybinds to human CD73” refers to an antibody that binds to soluble or cellbound human CD73 with a K_(D) of 10⁻⁷ M or less, such as approximatelyless than 10⁻⁸M, 10⁻⁹M or 10⁻¹⁰ M or even lower. An antibody that“cross-reacts with cynomolgus CD73” refers to an antibody that binds tocynomolgus CD73 with a K_(D) of 10⁻⁷ M or less, such as less than 10⁻⁸M, 10⁻⁹ M or 10⁻¹⁰ M or even lower. In certain embodiments, antibodiesthat do not cross-react with CD73 from a non-human species exhibitessentially undetectable binding against these proteins in standardbinding assays.

The term“k_(assoc)” or “k_(a)”, as used herein, is intended to refer tothe association rate constant of a particular antibody-antigeninteraction, whereas the term “k_(dis)” or “k_(d),” as used herein, isintended to refer to the dissociation rate constant of a particularantibody-antigen interaction. The term “K_(D)”, as used herein, isintended to refer to the equilibrium dissociation constant, which isobtained from the ratio of k_(d) to k_(a) (i.e., k_(d)/k_(a)) and isexpressed as a molar concentration (M). K_(D) values for antibodies canbe determined using methods well established in the art. A preferredmethod for determining the K_(D) of an antibody is by using surfaceplasmon resonance, preferably using a biosensor system such as aBiacore® surface plasmon resonance system or flow cytometry andScatchard analysis.

The term “EC50” in the context of an in vitro or in vivo assay using anantibody or antigen binding fragment thereof, refers to theconcentration of an antibody or an antigen-binding portion thereof thatinduces a response that is 50% of the maximal response, i.e., halfwaybetween the maximal response and the baseline.

A “rate of internalization” of an antibody or of a receptor, e.g., CD73,as mediated by the antibody, e.g., an anti-CD73 antibody, may berepresented, e.g., by T_(1/2) of internalization, e.g., as shown in theExamples. A rate of internalization of an anti-CD73 antibody may beenhanced or increased by at least 10%, 30%, 50%, 75%, 2 fold, 3 fold, 5fold or more, resulting in a reduction of the T_(1/2) by at least 10%,30%, 50%, 75%, 2 fold, 3 fold, 5 fold or more by changing the heavychain constant region of the antibody to a modified heavy chain constantregion, e.g., one that contains an IgG2 hinge and IgG2 CH1 domain. Forexample, instead of having a T_(1/2) of 10 minutes, a modified heavychain constant region may increase the rate of internalization andthereby reduce the T_(1/2) to 5 minutes (i.e., a two fold increase inrate of internalization or a two-fold decrease in T_(1/2)). “T_(1/2)” isdefined as the time at which half of the maximal internalization isachieved, as measured from the time the antibody is added to the cells.The maximal level of internalization can be the level of internalizationat the plateau of a graph representing the internalization plottedagainst antibody concentrations. A modified heavy chain constant regionmay increase the maximal level of internalization of an antibody by atleast 10%, 30%, 50%, 75%, 2 fold, 3 fold, 5 fold or more. Another way ofcomparing internalization efficacies of different antibodies, such as anantibody with, and the same antibody without, a modified heavy chainconstant region, is by comparing their level of internalization at agiven antibody concentration (e.g., 100 nM) or at a given time (e.g., 2minutes, 5 minutes, 10 minutes or 30 minutes). Comparing levels ofinternalization can also be done by comparing the EC₅₀ levels ofinternalization. The level of internalization of one antibody can bedefined relative to that of a given (reference) antibody, e.g., anantibody described herein, e.g., 11F11 or CD73.4-IgG2CS-IgG1 orCD73.4-IgG2CS-IgG1.1f, and, can be indicated as a percentage of thevalue obtained with the given (reference) antibody. The extent ofinternalization may be enhanced by at least 10%, 30%, 50%, 75%, 2 fold,3 fold, 5 fold or more, as compared by any one of these methods.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally-occurring.

A “polypeptide” refers to a chain comprising at least two consecutivelylinked amino acid residues, with no upper limit on the length of thechain. One or more amino acid residues in the protein may contain amodification such as, but not limited to, glycosylation, phosphorylationor a disulfide bond. A “protein” may comprise one or more polypeptides.

The term “nucleic acid molecule,” as used herein, is intended to includeDNA molecules and RNA molecules. A nucleic acid molecule may besingle-stranded or double-stranded, and may be cDNA.

Also provided are “conservative sequence modifications” of the sequencesset forth in SEQ ID NOs described herein, i.e., nucleotide and aminoacid sequence modifications which do not abrogate the binding of theantibody encoded by the nucleotide sequence or containing the amino acidsequence, to the antigen. Such conservative sequence modificationsinclude conservative nucleotide and amino acid substitutions, as wellas, nucleotide and amino acid additions and deletions. For example,modifications can be introduced into SEQ ID NOs described herein bystandard techniques known in the art, such as site-directed mutagenesisand PCR-mediated mutagenesis. Conservative sequence modificationsinclude conservative amino acid substitutions, in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine,tryptophan), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in an anti-CD73 antibody ispreferably replaced with another amino acid residue from the same sidechain family. Methods of identifying nucleotide and amino acidconservative substitutions that do not eliminate antigen binding arewell-known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187(1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burkset al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

Alternatively, in another embodiment, mutations can be introducedrandomly along all or part of an anti-CD73 antibody coding sequence,such as by saturation mutagenesis, and the resulting modified anti-CD73antibodies can be screened for improved binding activity.

For nucleic acids, the term “substantial homology” indicates that twonucleic acids, or designated sequences thereof, when optimally alignedand compared, are identical, with appropriate nucleotide insertions ordeletions, in at least about 80% of the nucleotides, usually at leastabout 90% to 95%, and more preferably at least about 98% to 99.5% of thenucleotides. Alternatively, substantial homology exists when thesegments will hybridize under selective hybridization conditions, to thecomplement of the strand.

For polypeptides, the term “substantial homology” indicates that twopolypeptides, or designated sequences thereof, when optimally alignedand compared, are identical, with appropriate amino acid insertions ordeletions, in at least about 80% of the amino acids, usually at leastabout 90% to 95%, and more preferably at least about 98% to 99.5% of theamino acids.

The percent identity between two sequences is a function of the numberof identical positions shared by the sequences when the sequences areoptimally aligned (i.e., % homology=# of identical positions/total # ofpositions×100), with optimal alignment determined taking into accountthe number of gaps, and the length of each gap, which need to beintroduced for optimal alignment of the two sequences. The comparison ofsequences and determination of percent identity between two sequencescan be accomplished using a mathematical algorithm, as described in thenon-limiting examples below.

The percent identity between two nucleotide sequences can be determinedusing the GAP program in the GCG software package (available athttp://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Thepercent identity between two nucleotide or amino acid sequences can alsobe determined using the algorithm of E. Meyers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percent identity betweentwo amino acid sequences can be determined using the Needleman andWunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

The nucleic acid and protein sequences described herein can further beused as a “query sequence” to perform a search against public databasesto, for example, identify related sequences. Such searches can beperformed using the NBLAST and)(BLAST programs (version 2.0) ofAltschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotidesearches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to the nucleicacid molecules described herein. BLAST protein searches can be performedwith the)(BLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to the protein molecules described herein. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g.,)(BLAST and NBLAST)can be used. See www.ncbi.nlm.nih.gov.

The nucleic acids may be present in whole cells, in a cell lysate, or ina partially purified or substantially pure form. A nucleic acid is“isolated” or “rendered substantially pure” when purified away fromother cellular components or other contaminants, e.g., other cellularnucleic acids (e.g., the other parts of the chromosome) or proteins, bystandard techniques, including alkaline/SDS treatment, CsCl banding,column chromatography, agarose gel electrophoresis and others well knownin the art. See, F. Ausubel, et al., ed. Current Protocols in MolecularBiology, Greene Publishing and Wiley Interscience, New York (1987).

Nucleic acids, e.g., cDNA, may be mutated, in accordance with standardtechniques to provide gene sequences. For coding sequences, thesemutations may affect amino acid sequence as desired. In particular, DNAsequences substantially homologous to or derived from native V, D, J,constant, switches and other such sequences described herein arecontemplated.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”) In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, also included are other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell that comprises a nucleic acidthat is not naturally present in the cell, and maybe a cell into which arecombinant expression vector has been introduced. It should beunderstood that such terms are intended to refer not only to theparticular subject cell but to the progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term “host cell” as used herein.

As used herein, the term “antigen” refers to any natural or syntheticimmunogenic substance, such as a protein, peptide, or hapten. An antigenmay be CD73 or a fragment thereof.

An “immune response” refers to a biological response within a vertebrateagainst foreign agents, which response protects the organism againstthese agents and diseases caused by them. An immune response is mediatedby the action of a cell of the immune system (for example, a Tlymphocyte, B lymphocyte, natural killer (NK) cell, macrophage,eosinophil, mast cell, dendritic cell or neutrophil) and solublemacromolecules produced by any of these cells or the liver (includingantibodies, cytokines, and complement) that results in selectivetargeting, binding to, damage to, destruction of, and/or eliminationfrom the vertebrate's body of invading pathogens, cells or tissuesinfected with pathogens, cancerous or other abnormal cells, or, in casesof autoimmunity or pathological inflammation, normal human cells ortissues. An immune response or reaction includes, e.g., activation orinhibition of a T cell, e.g., an effector T cell or a Th cell, such as aCD4+ or CD8+ T cell, or the inhibition of a Treg cell.

An “immunomodulator” or “immunoregulator” refers to an agent, e.g., acomponent of a signaling pathway, which may be involved in modulating,regulating, or modifying an immune response. “Modulating,” “regulating,”or “modifying” an immune response refers to any alteration in a cell ofthe immune system or in the activity of such cell (e.g., an effector Tcell). Such modulation includes stimulation or suppression of the immunesystem which may be manifested by an increase or decrease in the numberof various cell types, an increase or decrease in the activity of thesecells, or any other changes which can occur within the immune system.Both inhibitory and stimulatory immunomodulators have been identified,some of which may have enhanced function in a tumor microenvironment.The immunomodulator may be located on the surface of a T cell. An“immunomodulatory target” or “immunoregulatory target” is animmunomodulator that is targeted for binding by, and whose activity isaltered by the binding of, a substance, agent, moiety, compound ormolecule. Immunomodulatory targets include, for example, receptors onthe surface of a cell (“immunomodulatory receptors”) and receptorligands (“immunomodulatory ligands”).

An increased ability to stimulate an immune response, or the immunesystem, can result from an enhanced agonist activity of T cellcostimulatory receptors and/or an enhanced antagonist activity ofinhibitory receptors. An increased ability to stimulate an immuneresponse or the immune system may be reflected by a fold increase of theEC50 or maximal level of activity in an assay that measures an immuneresponse, e.g., an assay that measures changes in cytokine or chemokinerelease, cytolytic activity (determined directly on target cells orindirectly via detecting CD107a or granzymes) and proliferation. Theability to stimulate an immune response or the immune system activitymay be enhanced by at least 10%, 30%, 50%, 75%, 2 fold, 3 fold, 5 foldor more.

“Immunotherapy” refers to the treatment of a subject afflicted with, orat risk of contracting or suffering a recurrence of, a disease by amethod comprising inducing, enhancing, suppressing or otherwisemodifying an immune response.

“Immunostimulating therapy” or “immunostimulatory therapy” refers to atherapy that results in increasing (inducing or enhancing) an immuneresponse in a subject for, e.g., treating cancer.

“Potentiating an endogenous immune response” means increasing theeffectiveness or potency of an existing immune response in a subject.This increase in effectiveness and potency may be achieved, for example,by overcoming mechanisms that suppress the endogenous host immuneresponse or by stimulating mechanisms that enhance the endogenous hostimmune response.

“T effector” (“T_(eff)”) cells refers to T cells (e.g., CD4+ and CD8+ Tcells) with cytolytic activities as well as T helper (Th) cells, whichsecrete cytokines and activate and direct other immune cells, but doesnot include regulatory T cells (Treg cells).

As used herein, the term “linked” refers to the association of two ormore molecules. The linkage can be covalent or non-covalent. The linkagealso can be genetic (i.e., recombinantly fused). Such linkages can beachieved using a wide variety of art recognized techniques, such aschemical conjugation and recombinant protein production.

As used herein, “administering” refers to the physical introduction of acomposition comprising a therapeutic agent to a subject, using any ofthe various methods and delivery systems known to those skilled in theart. Preferred routes of administration for antibodies described hereininclude intravenous, intraperitoneal, intramuscular, subcutaneous,spinal or other parenteral routes of administration, for example byinjection or infusion. The phrase “parenteral administration” as usedherein means modes of administration other than enteral and topicaladministration, usually by injection, and includes, without limitation,intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal,intralymphatic, intralesional, intracapsular, intraorbital,intracardiac, intradermal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion, as well as in vivo electroporation.Alternatively, an antibody described herein can be administered via anon-parenteral route, such as a topical, epidermal or mucosal route ofadministration, for example, intranasally, orally, vaginally, rectally,sublingually or topically. Administering can also be performed, forexample, once, a plurality of times, and/or over one or more extendedperiods.

As used herein, the term “T cell-mediated response” refers to a responsemediated by T cells, including effector T cells (e.g., CD8⁺ cells) andhelper T cells (e.g., CD4⁺ cells). T cell mediated responses include,for example, T cell cytotoxicity and proliferation.

As used herein, the term “cytotoxic T lymphocyte (CTL) response” refersto an immune response induced by cytotoxic T cells. CTL responses aremediated primarily by CD8⁺ T cells.

As used herein, the terms “inhibits” or “blocks” (e.g., referring toinhibition/blocking of CD73 binding or activity) are usedinterchangeably and encompass both partial and completeinhibition/blocking.

As used herein, “cancer” refers a broad group of diseases characterizedby the uncontrolled growth of abnormal cells in the body. Unregulatedcell division may result in the formation of malignant tumors or cellsthat invade neighboring tissues and may metastasize to distant parts ofthe body through the lymphatic system or bloodstream.

The terms “treat,” “treating,” and “treatment,” as used herein, refer toany type of intervention or process performed on, or administering anactive agent to, the subject with the objective of reversing,alleviating, ameliorating, inhibiting, or slowing down or preventing theprogression, development, severity or recurrence of a symptom,complication, condition or biochemical indicia associated with adisease. Prophylaxis refers to administration to a subject who does nothave a disease, to prevent the disease from occurring or minimize itseffects if it does.

A “hematological malignancy” includes a lymphoma, leukemia, myeloma or alymphoid malignancy, as well as a cancer of the spleen and the lymphnodes. Exemplary lymphomas include both B cell lymphomas and T celllymphomas. B-cell lymphomas include both Hodgkin's lymphomas and mostnon-Hodgkin's lymphomas. Non-limiting examples of B cell lymphomasinclude diffuse large B-cell lymphoma, follicular lymphoma,mucosa-associated lymphatic tissue lymphoma, small cell lymphocyticlymphoma (overlaps with chronic lymphocytic leukemia), mantle celllymphoma (MCL), Burkitt's lymphoma, mediastinal large B cell lymphoma,Waldenström macroglobulinemia, nodal marginal zone B cell lymphoma,splenic marginal zone lymphoma, intravascular large B-cell lymphoma,primary effusion lymphoma, lymphomatoid granulomatosis. Non-limitingexamples of T cell lymphomas include extranodal T cell lymphoma,cutaneous T cell lymphomas, anaplastic large cell lymphoma, andangioimmunoblastic T cell lymphoma. Hematological malignancies alsoinclude leukemia, such as, but not limited to, secondary leukemia,chronic lymphocytic leukemia, acute myelogenous leukemia, chronicmyelogenous leukemia, and acute lymphoblastic leukemia. Hematologicalmalignancies further include myelomas, such as, but not limited to,multiple myeloma and smoldering multiple myeloma. Other hematologicaland/or B cell- or T-cell-associated cancers are encompassed by the termhematological malignancy.

The term “effective dose” or “effective dosage” is defined as an amountsufficient to achieve or at least partially achieve a desired effect. A“therapeutically effective amount” or “therapeutically effective dosage”of a drug or therapeutic agent is any amount of the drug that, when usedalone or in combination with another therapeutic agent, promotes diseaseregression evidenced by a decrease in severity of disease symptoms, anincrease in frequency and duration of disease symptom-free periods, or aprevention of impairment or disability due to the disease affliction. A“prophylactically effective amount” or a “prophylactically effectivedosage” of a drug is an amount of the drug that, when administered aloneor in combination with another therapeutic agent to a subject at risk ofdeveloping a disease or of suffering a recurrence of disease, inhibitsthe development or recurrence of the disease. The ability of atherapeutic or prophylactic agent to promote disease regression orinhibit the development or recurrence of the disease can be evaluatedusing a variety of methods known to the skilled practitioner, such as inhuman subjects during clinical trials, in animal model systemspredictive of efficacy in humans, or by assaying the activity of theagent in in vitro assays.

By way of example, an anti-cancer agent is a drug that slows cancerprogression or promotes cancer regression in a subject. In preferredembodiments, a therapeutically effective amount of the drug promotescancer regression to the point of eliminating the cancer. “Promotingcancer regression” means that administering an effective amount of thedrug, alone or in combination with an anti-neoplastic agent, results ina reduction in tumor growth or size, necrosis of the tumor, a decreasein severity of at least one disease symptom, an increase in frequencyand duration of disease symptom-free periods, a prevention of impairmentor disability due to the disease affliction, or otherwise ameliorationof disease symptoms in the patient. Pharmacological effectiveness refersto the ability of the drug to promote cancer regression in the patient.Physiological safety refers to an acceptably low level of toxicity, orother adverse physiological effects at the cellular, organ and/ororganism level (adverse effects) resulting from administration of thedrug.

By way of example for the treatment of tumors, a therapeuticallyeffective amount or dosage of the drug preferably inhibits cell growthor tumor growth by at least about 20%, more preferably by at least about40%, even more preferably by at least about 60%, and still morepreferably by at least about 80% relative to untreated subjects. In themost preferred embodiments, a therapeutically effective amount or dosageof the drug completely inhibits cell growth or tumor growth, i.e.,preferably inhibits cell growth or tumor growth by 100%. The ability ofa compound to inhibit tumor growth can be evaluated using the assaysdescribed infra. Alternatively, this property of a composition can beevaluated by examining the ability of the compound to inhibit cellgrowth, such inhibition can be measured in vitro by assays known to theskilled practitioner. In other preferred embodiments described herein,tumor regression may be observed and may continue for a period of atleast about 20 days, more preferably at least about 40 days, or evenmore preferably at least about 60 days.

The terms “patient” and “subject” refer to any human or non-human animalthat receives either prophylactic or therapeutic treatment. For example,the methods and compositions described herein can be used to treat asubject having cancer. The term “non-human animal” includes allvertebrates, e.g., mammals and non-mammals, such as non-human primates,sheep, dog, cow, chickens, amphibians, reptiles, etc.

Various aspects described herein are described in further detail in thefollowing subsections.

I. Anti-CD73 Antibodies

Described herein are antibodies, e.g., fully human antibodies, which arecharacterized by particular functional features or properties. Forexample, the antibodies specifically bind human CD73. Additionally,antibodies may cross react with CD73 from one or more non-humanprimates, such as cynomolgus CD73.

In addition to binding specifically to soluble and/or membrane boundhuman CD73, the antibodies described herein exhibit one or more of thefollowing functional properties:

(a) inhibition of CD73 enzymatic activity, resulting in a reduction ofadenosine produced;

(b) binding to cyno CD73;

(c) antibody mediated CD73 internalization into cells, e.g., tumorcells; and

(d) binding to a conformational epitope comprising amino acids 65-83 and157-172 of human CD73.

Preferably, anti-CD73 antibodies bind to human CD73 (monomeric ordimeric; isoform 1 or 2) with high affinity, for example, with a K_(D)of 10⁻⁷ M or less, 10⁻⁸ M or less, 10⁻⁹M or less, 10⁻¹⁰ M or less, 10⁻¹¹M or less, 10⁻¹² M or less, 10⁻¹² M to 10⁻⁷ M, 10⁻¹¹ M to 10⁻⁷ M, 10⁻¹⁰M to 10⁻⁷ M, 10⁻⁹ M to 10⁻⁷ M, or 10⁻¹⁰ M to 10⁻⁸M. In certainembodiments, an anti-CD73 antibody binds to soluble human CD73, e.g, asdetermined by BIACORE® SPR analysis, with a K_(D) of 10⁻⁷ M or less,10⁻⁸ M or less, 10⁻⁹M (1 nM) or less, 10⁻¹⁰ M or less, 10⁻¹² M to 10⁻⁷M, 10⁻¹¹ M to 10⁻⁷ M, 10⁻¹⁰ M to 10⁻⁷ M, 10⁻⁹ M to 10⁻⁷ M, 10⁻⁸ M to10⁻⁷ M or 10⁻¹⁰ M to 10⁻⁸ M. In certain embodiments, an anti-CD73antibody binds to bound (e.g., cell membrane bound, e.g., Calu6 cells)human CD73, e.g., as determined as further described herein, with anEC50 of less than 1 nM. In certain embodiments, an anti-CD73 antibodybinds to bound human CD73, e.g., cell membrane bound human CD73, e.g.,as determined by flow cytometry and Scatchard plot, with a K_(D) of 10⁻⁷M or less, 10⁻⁸ M or less, 10⁻⁹M (1 nM) or less, 10⁻¹⁰ M or less, 10⁻¹²M to 10⁻⁷ M, 10⁻¹¹ M to 10⁻⁸ M, 10⁻¹⁰ M to 10⁻⁸ M, 10⁻⁹ M to 10⁻⁸ M,10⁻¹¹ M to 10⁻⁹ M, 10⁻¹⁰ M to 10⁻⁸ M, or 10⁻¹⁰ M to 10⁻⁹ M. In certainembodiments, an anti-CD73 antibody binds to soluble human CD73 with aK_(D) of 10⁻⁷ M or less, 10⁻⁸ M or less, 10⁻⁹M (1 nM) or less, 10⁻¹⁰ Mor less, 10⁻¹² M to 10⁻⁷ M, 10⁻¹¹ M to 10⁻⁷ M, 10⁻¹⁰ M to 10⁻⁷ M, 10⁻⁹Mto 10⁻⁷ M, 10⁻¹⁰ M to 10⁻⁸ M, or 10⁻⁸M to 10⁻⁷ M, and to bound humanCD73, e.g., cell membrane bound human CD73, with a K_(D) or EC₅₀ of 10⁻⁷M or less, 10⁻⁸ M or less, 10⁻⁹M (1 nM) or less, 10⁻¹⁰ M or less, 10⁻¹²M to 10⁻⁷ M, 10⁻¹¹M to 10⁻⁸ M, 10⁻¹⁰ M to 10⁻⁸ M, 10⁻⁹M to 10⁻⁸M, 10⁻¹¹M to 10⁻⁹ M, or 10⁻¹⁰ M to 10⁻⁹ M.

In certain embodiments, an anti-CD73 antibody binds to cyno CD73 withhigh affinity, e.g., it binds to a CHO cell expressing cyno CD73 with anEC50 of 0.1 nM to 10 nM, such as an EC50 of less than 1 nM, asdetermined, e.g., as further described herein.

In certain embodiments, anti-CD73 antibodies described herein also bindto cynomolgus CD73, e.g., bind to membrane bound cynomolgus CD73, e.g,to a CHO cell expressing cyno CD73 with an EC₅₀ of 100 nM or less, 10 nMor less, 1 nM or less, 100 nM to 0.01 nM, 100 nM to 0.1 nM, 100 nM to 1nM, or 10 nM to 0.1 nM, as measured, e.g., in the Examples.

In certain embodiments, anti-CD73 antibodies are at least 90%, 95%, 98%,or 99% monomeric, as determined, e.g., by SEC. Anti-CD73 antibodies mayalso have biophysical characteristics that are similar to, or within therange of, those of the antibodies described herein.

In certain embodiments, anti-CD73 antibodies inhibit the enzymaticactivity of human and/or cyno CD73, e.g., as determined in CD73 beadbound assays, or as determined in cells, e.g., Calu6, SKMEL24 or H292cells, or as determined in an in vivo assay, e.g., a xenograft tumormodel, e.g., as further described in the Examples. Anti-CD73 antibodiesmay have inhibitory activities that are at least similar to, or withinthe range of, those of the antibodies described herein. For example,anti-CD73 antibodies may inhibit human CD73 (e.g., CD73 bound to asolid) enzymatic activity (adenosine production) with an EC₅₀ of lessthan 10 nM or less than 5 nM or in the range of 1 to 10 nM or 5 to 10nM. Anti-CD73 antibodies may inhibit the activity of human CD73 oncells, e.g., Calu6 cells with an EC₅₀ of less than 10 nM or less than 1nM or in the range of 0.1 to 10 nM, 0.1 to 1 nM or 0.1 to 0.5 nM.

In certain embodiments, anti-CD73 antibodies are internalized (andmediate CD73 internalization) by a cell to which it binds as determined,e.g., in a high content internalization assay or by FACS or flowcytometry, as further described in the Examples. Anti-CD73 antibodiesmay have internalization characteristics (EC50, T_(1/2) and Ymax), andtime to plateau that are at least similar to, or within the range of,those of the antibodies described in the Examples. In certainembodiments, an anti-CD73 antibody has a T_(1/2) of internalization thatis less than 1 hour, such as less than 30 minutes, less than 15 minutes,less than 12 minutes, less than 10 minutes, less than 7 minutes or evenless than 5 minutes in one or more cell lines, e.g, those set forth inthe Examples, as determined, e.g., in a high content internalizationassay (described in Example 6A). In certain embodiments, an anti-CD73antibody reaches maximal anti-CD73 antibody mediated internalizationwithin 10 hours or less, 6 hours or less, 5 hours or less, 4 hours orless, 3 hours or less, 2 hours or less, 1 hour or less, e.g., in therange of 10 minutes to 10 hours, 10 minutes to 6 hours, 1 hour to 10hours or 1 hour to 6 hours, as determined, e.g., using a high contentinternalization assay, as described, e.g., in Example 6A, or using flowcytometry, as described, e.g., in Example 6B. The maximal level ofanti-CD73 antibody mediated internalization of CD73 may be at least 50%,at least 60%, at least 70%, at least 80%, at least 90% or more,depending on the cell type. For example, the EC50 of anti-CD73 antibodymediated internalization of CD73 in Calu6 cells, as measured in the highcontent internalization assay described in the Examples, may be lessthan 10 nM, e.g., from 0.1 to 10 nM or 1 to 10 nM or 1 to 5 nM and aYmax of at least 90% or at least 95%.

Anti-CD73 antibodies, e.g., antibodies having an IgG2 hinge, IgG2 CH1domain, or IgG2 hinge and IgG2 CH1 domain, may mediate the followingCD73 internalization characteristics as measured in a high contentinternalization assay, e.g., as described in Example 6A:

-   -   EC50 of 10 nM or less, 5 nM or less, 1 nM or less, or 0.1 to 10        nM or 0.1 to 1 nM; a Ymax (maximal percentage of        internalization) of at least 90%, 95% or 98% in Calu6 cells and        a T_(1/2) of less than 30 minutes or less than 10 minutes in        Calu6 cells;    -   A T_(1/2) of less than 30 minutes or less than 10 minutes in        human cells, e.g., Calu6 cells, HCC44 cells, H2030 cells, H2228        cells, HCC15 cells, SKLU1 cells, SKMES1 cells or SW900 cells.

Anti-CD73 antibodies, e.g., antibodies having an IgG2 hinge, IgG2 CH1domain, or IgG2 hinge and IgG2 CH1 domain, may mediate the followingCD73 internalization characteristics as measured by flow cytometry,e.g., as described in Example 6B:

-   -   A T_(1/2) of 1 hour or less and a Ymax of at least 70% in Calu6        cells;    -   A T_(1/2) of 30 minutes or less and a Ymax of at least 70% in        NCI-H292 cells;    -   A T_(1/2) of 2 hours or less and a Ymax of at least 30% in SNUC1        cells; and/or    -   A T_(1/2) of 30 minutes or less and a Ymax of at least 60% in        NCI-H1437 cells.

In certain embodiments, an anti-CD73 antibody is a bin1 antibody, i.e.,it competes for binding to human CD73 with 11F11, but not with 4C3.

In certain embodiments, anti-CD73 antibodies bind to an epitope, e.g., aconformational epitope in the N-terminal portion of human CD73, e.g., anepitope located within amino acids 65-83 of human CD73 (SEQ ID NO:96),as determined, e.g., by HDX-MS, as further described in the Examples. Incertain embodiments, anti-CD73 antibodies bind to amino acids 157-172 ofhuman CD73 (SEQ ID NO: 97), or to an epitope located within amino acids157-172, of human CD73 (SEQ ID NO: 97), as determined, e.g., by HDX-MS.Alternatively, anti-CD73 antibodies bind to an epitope, e.g., adiscontinuous epitope in the N-terminal portion of human CD73, asdetermined, e.g., by HDX-MS.

In certain embodiments, anti-CD73 antibodies bind to amino acids 65 to83 and amino acids 157-172 of human CD73, or to an epitope within aminoacids 65 to 83 and amino acids 157-172, of human CD73 isoform 1 or 2,i.e., amino acid sequences FTKVQQIRRAEPNVLLLDA (SEQ ID NO: 96) andLYLPYKVLPVGDEVVG (SEQ ID NO: 97), as determined by, e.g., HDX-MS. Incertain embodiments, the anti-CD73 antibodies bind to all or a portionof amino acids 65 to 83 and amino acids 157-172 of human CD73, asdetermined by, e.g., HDX-MS. In certain embodiments, anti-CD73antibodies bind to both glycosylated and unglycosylated human CD73. Incertain embodiments, anti-CD73 antibodies bind only to glycosylated CD73and not to unglycosylated CD73.

Anti-CD73 antibodies may compete for binding to CD73 with (or inhibitbinding of) anti-CD73 antibodies comprising CDRs or variable regionsdescribed herein, e.g., those of CD73.4-1, CD73.4-2, CD73.3, 11F11-1,11F11-2, 4C3-1, 4C3-2, 4C3-3, 4D4, 10D2-1, 10D2-2, 11A6, 24H2, 5F8-1,5F8-2, 6E11 and/or 7A11. In certain embodiments, anti-CD73 antibodiesinhibit binding of CD73.4-1, CD73.4-2, CD73.3, 11F11, 4C3, 4D4, 10D2,11A6, 24H2, 5F8, 6E11 and/or 7A11 to human CD73 by at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90% or by 100%. In certain embodiments,CD73.4-1, CD73.4-2, CD73.3, 11F11-1, 11F11-2, 4C3-1, 4C3-2, 4C3-3, 4D4,10D2-1, 10D2-2, 11A6, 24H2, 5F8-1, 5F8-2, 6E11 and/or 7A11 inhibitbinding of anti-CD73 antibodies to human CD73 by at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90% or by 100%. In certain embodiments,anti-CD73 antibodies inhibit binding of CD73.4-1, CD73.4-2, CD73.3,11F11-1, 11F11-2, 4C3-1, 4C3-2, 4C3-3, 4D4, 10D2-1, 10D2-2, 11A6, 24H2,5F8-1, 5F8-2, 6E11 and/or 7A11 to human CD73 by at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90% or by 100% and CD73.4-1, CD73.4-2, CD73.3,11F11-1, 11F11-2, 4C3-1, 4C3-2, 4C3-3, 4D4, 10D2-1, 10D2-2, 11A6, 24H2,5F8-1, 5F8-2, 6E11 and/or 7A11 inhibit binding of the anti-CD73antibodies to human CD73 by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90% or by 100% (e.g., compete in both directions). Competitionexperiments may be performed, e.g., as further described herein, e.g.,in the Examples.

In certain embodiments, anti-CD73 antibodies inhibit CD73 enzymaticactivity and/or are internalized in cells without requiring multivalentcross-linking, as determined, e.g., by the lack of requirement of FcRbinding.

In certain embodiments, anti-CD73 antibodies have 1, 2, 3, 4, 5, 6, 7,8, 9, 10, or 11 of the features listed in Table 3.

Table 3: Potential features of anti-CD73 antibodies

-   -   (1) binding to human CD73, e.g., bead bound human monomeric and        dimeric human CD73 isoform 1 and 2, e.g., with a K_(D) of 10 nM        or less (e.g., 0.01 nM to 10 nM), e.g., as measured by BIACORE®        SPR analysis;    -   (2) binding to membrane bound human CD73, e.g., with an EC₅₀ of        1 nM or less (e.g., 0.01 nM to 1 nM);    -   (3) binding to cynomolgus CD73, e.g., binding to membrane bound        cynomolgus CD73, e.g, with an EC₅₀ of 10 nM or less (e.g., 0.01        nM to 10 nM);    -   (4) inhibition of human CD73 enzymatic activity, e.g., with an        EC50 of 10 nM or less;    -   (5) inhibition of cyno CD73 enzymatic activity, e.g., with an        EC50 of 10 nM or less;    -   (6) inhibition of endogenous (cellular) human CD73 enzymatic        activity in Calu6 cells with an EC50 of 10 nM or less;    -   (7) inhibition of human CD73 enzymatic activity in vivo;    -   (8) internalization, e.g., antibody mediated (or dependent) CD73        internalization, into cells, e.g., with a T_(1/2) of less than 1        hour, 30 minutes or 10 minutes and/or a Ymax of at least 70%,        80% or 90%;    -   (9) binding to a conformational epitope on human CD73, e.g., a        discontinuous epitope within the amino acid sequence (SEQ ID        NO: 1) which includes all or a portion of amino acid residues        FTKVQQIRRAEPNVLLLDA (SEQ ID NO: 96) and/or LYLPYKVLPVGDEVVG (SEQ        ID NO: 97);    -   (10) competing in either direction or both directions for        binding to human CD73 with CD73.4-1, CD73.4-2, CD73.3, 11F11-1,        11F11-2, 4C3-1, 4C3-2, 4C3-3, 4D4, 10D2-1, 10D2-2, 11A6, 24H2,        5F8-1, 5F8-2, 6E11 and/or 7A11; and    -   (11) interacting with human CD73 in a similar pattern as CD73.4,        as determined by X-ray crystallography.

An antibody activity that exhibits one or more of these functionalproperties (e.g., biochemical, immunochemical, cellular, physiologicalor other biological activities, or the like) as determined according tomethodologies known to the art and described herein, will be understoodto relate to a statistically significant difference in the particularactivity relative to that seen in the absence of the antibody (e.g., orwhen a control antibody of irrelevant specificity is present). Incertain embodiments, an anti-CD73 antibody disclosed herein decreases ameasured parameter (e.g., tumor volume, tumor metastasis, adenosinelevels, cAMP levels) by at least 10% of the measured parameter, morepreferably by at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, and incertain preferred embodiments, by greater than 92%, 94%, 95%, 97%, 98%or 99%. Conversely, an anti-CD73 antibody disclosed herein increases ameasured parameter by at least 10%, such as by at least 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, 100% (i.e. 2 fold), 3 fold, 5 fold, or 10fold.

Standard assays to evaluate the binding ability of the antibodies towardCD73 of various species are known in the art, including for example,ELISAs, Western blots, and RIAs. Suitable assays are described in detailin the Examples. The binding kinetics (e.g., binding affinity) of theantibodies also can be assessed by standard assays known in the art,such as by BIACORE® SPR analysis. Assays to evaluate the effects of theantibodies on functional properties of CD73 (e.g., adenosine production,tumor growth and metastasis, T cell inhibition) are described in furtherdetail infra and in the Examples.

In certain embodiments, anti-CD73 antibodies are not native antibodiesor are not naturally-occurring antibodies. For example, anti-CD73antibodies have post-translational modifications that are different fromthose of antibodies that are naturally occurring, such as by havingmore, less or a different type of post-translational modification.

In certain embodiments, anti-CD73 antibodies stimulate Teff (T effector)function and/or reduce Treg function, e.g., by removing CD73 from the Tcell surface and/or by inhibiting its enzymatic activity.

In certain embodiments, anti-CD3 antibodies comprise at least an IgG2hinge, and optionally also an IgG2 CH1 domain or fragment or derivativeof the hinge and/or CH1 domain and the antibody form A (see, e.g., Allenet al. (2009) Biochemistry 48:3755). In certain embodiments, anti-CD3antibodies comprise at least an IgG2 hinge, and optionally also an IgG2CH1 domain or fragment or derivative of the hinge and/or CH1 domain andthe antibody has adopted form B (see, e.g., Allen et al. (2009)Biochemistry 48:3755). In certain embodiments a composition comprises amixture of anti-CD73 antibodies with form A and anti-CD73 antibodieswith form B.

Provided herein are anti-human CD73 antibodies that (i) comprise avariable region that binds to a region on human CD73 that is similar tothat bound by 11F11, but does not bind to a region that is similar tothat bound by 4C3 (i.e., is a bin1 antibody); (ii) bind to monomeric anddimeric human CD73 with a Kd of 10 nM or less; (iii) inhibit theenzymatic activity (conversion of AMP to adenosine) of human CD73, e.g.,on cells, e.g., Calu6 cells, with an EC₅₀ of less than 10 nM; and (iv)mediate antibody dependent CD73 internalization in cells, e.g., with aT_(1/2) of 1 hour or less (or 30 minutes or less, or 10 minutes orless), a Ymax of 50% or more (or 60% or more, 70% or more, 80% or moreor 90% or more) in human cells, e.g., Calu6 cells, H2228 cells, HCC15cells H2030 cells, SNUC1 cells. In certain embodiments, the antibodiescomprise an IgG2 hinge or an IgG2 hinge and IgG2 CH1 domain. Providedherein are anti-human CD73 antibodies that (i) comprise a variableregion that binds to a region on human CD73 that is similar to thatbound by 11F11, but does not bind to a region that is similar to thatbound by 4C3 (i.e., is a bin1 antibody); (ii) bind to monomeric anddimeric human CD73 with a Kd of 10 nM or less, as determined by SPR(Biacore); (iii) inhibit the enzymatic activity (conversion of AMP toadenosine) of human CD73, e.g., on cells, e.g., Calu6 cells, with anEC₅₀ of less than 10 nM; and (iv) mediate antibody dependent CD73internalization in cells, e.g., with a T_(1/2) of 30 minutes or less, aYmax of 80% or more in human Calu6, H2228, HCC15 or H2030 cells, asdetermined using the high content internalization assay described inExample 6A.

In preferred embodiments, an anti-CD73 antibody described herein is notsignificantly toxic. For example, an anti-CD73 antibody is notsignificantly toxic to an organ of a human, e.g., one or more of theliver, kidney, brain, lungs, and heart, as determined, e.g., in clinicaltrials. In certain embodiments, an anti-CD73 antibody does notsignificantly trigger an undesirable immune response, e.g., autoimmunityor inflammation.

II. Exemplary Anti-CD73 Antibodies Variable Regions of Anti-CD73Antibodies

Particular antibodies described herein are antibodies, e.g., monoclonalantibodies, having the CDR and/or variable region sequences ofantibodies 11F11-1, 11F11-2, 4C3-1, 4C3-2, 4C3-3, 4D4, 10D2-1, 10D2-2,11A6, 24H2, 5F8-1, 5F8-2, 6E11, 7A11, CD73.3-1, -2 or -3, CD73.4-1 and-2, CD73.4-2, CD73.5-1 and -2, CD73.6-1 and -2, CD73.7-1 and -2,CD73.8-1 and -2, CD73.9-1 and -2, CD73.10-1 and -2 and CD73.11, as wellas antibodies having at least 80% identity (e.g., at least 85%, at least90%, at least 95%, or at least 99% identity) to their variable region orCDR sequences. Table 4 sets forth the SEQ ID NOs of the CDRs of the VHand VL regions of each antibody, as well as that of the VH and VLregions. As further described in the Examples, certain heavy chains canexist with more than one light chain, and the SEQ ID NOs of thealternate light chains are also provided in the Table below.

TABLE 4 VH VL CDR1 CDR2 CDR3 VH CDR1 CDR2 CDR3 VL 11F11-1 5 6 7 4 9 1011 8 11F11-2 5 6 7 4 13 14 15 12 4C3-1 17 18 19 16 21 22 23 20 4C3-2 1718 19 16 25 26 27 24 4C3-3 17 18 19 16 29 30 31 28 4D4-1 33 34 35 32 3738 39 36 10D2-1 41 42 43 40 45 46 47 44 10D2-2 41 42 43 40 49 50 51 4811A6-1 53 54 55 52 57 58 59 56 24H2-1 61 62 63 60 65 66 67 64 5F8-1 6970 71 68 73 74 75 72 5F8-2 69 70 71 68 77 78 79 76 5F8-3 69 70 71 68 239240 241 238 6E11-1 81 82 83 80 85 86 87 84 7A11-1 89 90 91 88 93 94 9592 73.3 17 18 19 170 21 22 23 20 73.4-1 5 6 7 135 9 10 11 8 73.4-2 5 6 7135 13 14 15 12 73.5-1 5 6 7 171 9 10 11 8 73.5-2 5 6 7 171 13 14 15 1273.6-1 5 6 7 172 9 10 11 8 73.6-2 5 6 7 172 13 14 15 12 73.7-1 5 6 7 1739 10 11 8 73.7-2 5 6 7 173 13 14 15 12 73.8-1 5 6 7 174 9 10 11 8 73.8-25 6 7 174 13 14 15 12 73.9-1 5 6 7 175 9 10 11 8 73.9-2 5 6 7 175 13 1415 12 73.10-1 5 6 7 176 9 10 11 8 73.10-2 5 6 7 176 13 14 15 12 73.11 3334 35 177 37 38 39 36

Provided herein are isolated antibodies, or antigen binding portionthereof, comprising heavy and light chain variable regions, wherein theheavy chain variable region comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 4, 16, 32, 40, 52, 60, 68, 80,88, 135, and 170-177.

Also provided are isolated antibodies, or antigen binding portionsthereof, comprising heavy and light chain variable regions, wherein thelight chain variable region comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 8, 12, 20, 24, 28, 36, 44, 48,56, 64, 72, 76, 84, 92 and 238.

Provided herein are isolated antibodies, or antigen-binding portionthereof, comprising:

(a) heavy and light chain variable region sequences comprising SEQ IDNOs: 135 and 8, respectively;

(b) heavy and light chain variable region sequences comprising SEQ IDNOs: 135 and 12, respectively;

(c) heavy and light chain variable region sequences comprising SEQ IDNOs: 4 and 8, respectively;

(d) heavy and light chain variable region sequences comprising SEQ IDNOs: 4 and 12, respectively;

(e) heavy and light chain variable region sequences comprising SEQ IDNOs: 16 and 20, respectively;

(f) heavy and light chain variable region sequences comprising SEQ IDNOs: 16 and 24, respectively;

(g) heavy and light chain variable region sequences comprising SEQ IDNOs: 16 and 28, respectively;

(h) heavy and light chain variable region sequences comprising SEQ IDNOs: 32 and 36, respectively;

(i) heavy and light chain variable region sequences comprising SEQ IDNOs: 40 and 44, respectively;

(j) heavy and light chain variable region sequences comprising SEQ IDNOs: 40 and 48, respectively;

(k) heavy and light chain variable region sequences comprising SEQ IDNOs: 52 and 56, respectively;

(l) heavy and light chain variable region sequences comprising SEQ IDNOs: 60 and 64, respectively;

(m) heavy and light chain variable region sequences comprising SEQ IDNOs: 68 and 72, respectively;

(n) heavy and light chain variable region sequences comprising SEQ IDNOs: 68 and 76, respectively;

(o) heavy and light chain variable region sequences comprising SEQ IDNOs: 68 and 238, respectively;

(p) heavy and light chain variable region sequences comprising SEQ IDNOs: 80 and 84, respectively;

(q) heavy and light chain variable region sequences comprising SEQ IDNOs: 88 and 92, respectively;

(r) heavy and light chain variable region sequences comprising SEQ IDNOs: 170 and 20, respectively;

(s) heavy and light chain variable region sequences comprising SEQ IDNOs: 170 and 24, respectively;

(t) heavy and light chain variable region sequences comprising SEQ IDNOs: 170 and 28, respectively;

(u) heavy and light chain variable region sequences comprising SEQ IDNOs: 171 and 8, respectively;

(v) heavy and light chain variable region sequences comprising SEQ IDNOs: 171 and 12, respectively;

(w) heavy and light chain variable region sequences comprising SEQ IDNOs: 172 and 8, respectively;

(x) heavy and light chain variable region sequences comprising SEQ IDNOs: 172 and 12, respectively;

(y) heavy and light chain variable region sequences comprising SEQ IDNOs: 173 and 8, respectively;

(z) heavy and light chain variable region sequences comprising SEQ IDNOs: 173 and 12, respectively;

(a2) heavy and light chain variable region sequences comprising SEQ IDNOs: 174 and 8, respectively;

(b2) heavy and light chain variable region sequences comprising SEQ IDNOs: 174 and 12, respectively;

(c2) heavy and light chain variable region sequences comprising SEQ IDNOs: 175 and 8, respectively;

(d2) heavy and light chain variable region sequences comprising SEQ IDNOs: 175 and 12, respectively;

(e2) heavy and light chain variable region sequences comprising SEQ IDNOs: 176 and 8, respectively;

(f2) heavy and light chain variable region sequences comprising SEQ IDNOs: 176 and 12, respectively; or

(g2) heavy and light chain variable region sequences comprising SEQ IDNOs: 177 and 36, respectively.

Anti-CD73 antibodies may comprise the heavy and light chain CDR1s, CDR2sand CDR3s of anti-CD73 antibodies described herein, e.g., CD73.4-1,CD73.4-2, CD73.3, 11F11-1, 11F11-2, 11F11, 4C3-1, 4C3-2, 4C3-3, 4D4,10D2-1, 10D2-2, 11A6, 24H2, 5F8-1, 5F8-2, 5F8-3, 6E11 and 7A11, orcombinations thereof.

Given that each of these antibodies binds to CD73 and thatantigen-binding specificity is provided primarily by the CDR1, 2 and 3regions, the V_(H) CDR1, 2 and 3 sequences and V_(L) CDR1, 2 and 3sequences can be “mixed and matched” (i.e., CDRs from differentantibodies can be mixed and match, although each antibody must contain aV_(H) CDR1, 2 and 3 and a V_(L) CDR1, 2 and 3) to create other anti-CD73binding molecules described herein. CD73 binding of such “mixed andmatched” antibodies can be tested using the binding assays describedabove and in the Examples (e.g., ELISAs). Preferably, when V_(H) CDRsequences are mixed and matched, the CDR1, CDR2 and/or CDR3 sequencefrom a particular V_(H) sequence is replaced with a structurally similarCDR sequence(s). Likewise, when V_(L) CDR sequences are mixed andmatched, the CDR1, CDR2 and/or CDR3 sequence from a particular V_(L)sequence preferably is replaced with a structurally similar CDRsequence(s). It will be readily apparent to the ordinarily skilledartisan that novel V_(H) and V_(L) sequences can be created bysubstituting one or more V_(H) and/or V_(L) CDR region sequences withstructurally similar sequences from the CDR sequences disclosed hereinfor monoclonal antibodies CD73.4-1, CD73.4-2, 11F11-1, 11F11-2, 4C3-1,4C3-2, 4C3-3, 4D4, 10D2-1, 10D2-2, 11A6, 24H2, 5F8-1, 5F8-2, 6E11 and/or7A11. “Mixed and matched” antibodies having binding affinity,bioactivity and/or other properties equivalent or superior to thespecific antibodies disclosed herein may be selected for use in themethods of the present invention.

Provided herein are isolated antibodies, or antigen binding portionthereof comprising:

(a) a heavy chain variable region CDR1 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 5, 17, 33, 41, 53, 61,69, 81, and 89;

(b) a heavy chain variable region CDR2 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 6, 18, 34, 42, 54, 62,70, 82, and 90;

(c) a heavy chain variable region CDR3 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 7, 19, 35, 43, 55, 63,71, 83, and 91;

(d) a light chain variable region CDR1 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 9, 13, 21, 25, 29, 37,45, 49, 57, 65, 73, 77, 85, and 93;

(e) a light chain variable region CDR2 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 10, 14, 22, 26, 30,38, 46, 50, 58, 66, 74, 78, 86, and 94; and

(f) a light chain variable region CDR3 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 11, 15, 23, 27, 31,39, 47, 51, 59, 67, 75, 79, 87, and 95;

wherein the antibody specifically binds to human CD73.

In certain embodiments, the antibody comprises heavy and light chainvariable regions, wherein the heavy chain variable region CDR1, CDR2,and CDR3 regions comprise SEQ ID NOs: 5-7; 17-19; 33-35; 41-43; 53-55;61-63; 69-71; 81-83; or 89-91;

wherein the antibody specifically binds to human CD73.

In certain embodiments, the antibody comprises heavy and light chainvariable regions, wherein the light chain variable region CDR1, CDR2,and CDR3 regions comprise:

-   -   (a) SEQ ID NOs: 9-11; 13-15; 21-23; 25-27; 29-31; 37-39; 45-47;        49-51; 57-59; 65-67; 73-75; 77-79; 85-87; or 93-95;

wherein the antibody specifically binds to human CD73.

In certain embodiments, the antibody comprises heavy and light chainvariable regions, wherein:

(a) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 5-7, respectively, and the light chain variable region CDR1,CDR2, and CDR3 comprises SEQ ID NOs: 9-11, respectively;

(b) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 5-7, respectively, and the light chain variable region CDR1,CDR2, and CDR3 comprises SEQ ID NOs: 13-15, respectively;

(c) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 17-19, respectively, and the light chain variable region CDR1,CDR2, and CDR3 comprises SEQ ID NOs: 21-23, respectively;

(d) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 17-19, respectively, and the light chain variable region CDR1,CDR2, and CDR3 comprises SEQ ID NOs: 25-27, respectively;

(e) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 17-19, respectively, and the light chain variable region CDR1,CDR2, and CDR3 comprises SEQ ID NOs: 29-31, respectively;

(f) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 33-35, respectively, and the light chain variable region CDR1,CDR2, and CDR3 comprises SEQ ID NOs: 37-39, respectively;

(g) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 41-43, respectively, and the light chain variable region CDR1,CDR2, and CDR3 comprises SEQ ID NOs: 45-47, respectively;

(h) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 41-43, respectively, and the light chain variable region CDR1,CDR2, and CDR3 comprises SEQ ID NOs: 49-51, respectively;

(i) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 53-55, respectively, and the light chain variable region CDR1,CDR2, and CDR3 comprises SEQ ID NOs: 57-59, respectively;

(j) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 61-63, respectively, and the light chain variable region CDR1,CDR2, and CDR3 comprises SEQ ID NOs: 65-67, respectively;

(k) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 69-71, respectively, and the light chain variable region CDR1,CDR2, and CDR3 comprises SEQ ID NOs: 73-75, respectively;

(l) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 69-71, respectively, and the light chain variable region CDR1,CDR2, and CDR3 comprises SEQ ID NOs: 77-79, respectively;

(m) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 81-83, respectively, and the light chain variable region CDR1,CDR2, and CDR3 comprises SEQ ID NOs: 85-87, respectively; or

(n) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 89-91, respectively, and the light chain variable region CDR1,CDR2, and CDR3 comprises SEQ ID NOs: 93-95, respectively;

wherein the antibody specifically binds to human CD73, and optionallyhas one or more of the characteristics listed in Table 3, e.g., theability to inhibit dephosphorylation of AMP and to mediate receptordependent CD73 internalization.

In certain embodiments, the antibody comprises heavy and light chainvariable regions, wherein:

(a) the heavy chain variable region CDR1, CDR2, and CDR3 consist of SEQID NOs: 5-7, respectively, and the light chain variable region CDR1,CDR2, and CDR3 consist of SEQ ID NOs: 9-11, respectively;

(b) the heavy chain variable region CDR1, CDR2, and CDR3 consist of SEQID NOs: 5-7, respectively, and the light chain variable region CDR1,CDR2, and CDR3 consist of SEQ ID NOs: 13-15, respectively;

(c) the heavy chain variable region CDR1, CDR2, and CDR3 consist of SEQID NOs: 17-19, respectively, and the light chain variable region CDR1,CDR2, and CDR3 consist of SEQ ID NOs: 21-23, respectively;

(d) the heavy chain variable region CDR1, CDR2, and CDR3 consist of SEQID NOs: 17-19, respectively, and the light chain variable region CDR1,CDR2, and CDR3 consist of SEQ ID NOs: 25-27, respectively;

(e) the heavy chain variable region CDR1, CDR2, and CDR3 consist of SEQID NOs: 17-19, respectively, and the light chain variable region CDR1,CDR2, and CDR3 consist of SEQ ID NOs: 29-31, respectively;

(f) the heavy chain variable region CDR1, CDR2, and CDR3 consist of SEQID NOs: 33-35, respectively, and the light chain variable region CDR1,CDR2, and CDR3 consist of SEQ ID NOs: 37-39, respectively;

(g) the heavy chain variable region CDR1, CDR2, and CDR3 consist of SEQID NOs: 41-43, respectively, and the light chain variable region CDR1,CDR2, and CDR3 consist of SEQ ID NOs: 45-47, respectively;

(h) the heavy chain variable region CDR1, CDR2, and CDR3 consist of SEQID NOs: 41-43, respectively, and the light chain variable region CDR1,CDR2, and CDR3 consist of SEQ ID NOs: 49-51, respectively;

(i) the heavy chain variable region CDR1, CDR2, and CDR3 consist of SEQID NOs: 53-55, respectively, and the light chain variable region CDR1,CDR2, and CDR3 consist of SEQ ID NOs: 57-59, respectively;

(j) the heavy chain variable region CDR1, CDR2, and CDR3 consist of SEQID NOs: 61-63, respectively, and the light chain variable region CDR1,CDR2, and CDR3 consist of SEQ ID NOs: 65-67, respectively;

(k) the heavy chain variable region CDR1, CDR2, and CDR3 consist of SEQID NOs: 69-71, respectively, and the light chain variable region CDR1,CDR2, and CDR3 consist of SEQ ID NOs: 73-75, respectively;

(l) the heavy chain variable region CDR1, CDR2, and CDR3 consist of SEQID NOs: 69-71, respectively, and the light chain variable region CDR1,CDR2, and CDR3 consist of SEQ ID NOs: 77-79, respectively;

(m) the heavy chain variable region CDR1, CDR2, and CDR3 consist of SEQID NOs: 81-83, respectively, and the light chain variable region CDR1,CDR2, and CDR3 consist of SEQ ID NOs: 85-87, respectively; or

(n) the heavy chain variable region CDR1, CDR2, and CDR3 consist of SEQID NOs: 89-91, respectively, and the light chain variable region CDR1,CDR2, and CDR3 consist of SEQ ID NOs: 93-95, respectively;

wherein the antibody specifically binds to human CD73, and optionallyhas one or more of the characteristics listed in Table 3, e.g., theability to inhibit dephosphorylation of AMP and to mediate receptordependent CD73 internalization.

Heavy Chain Constant Domains of Anti-CD73 Antibodies

The heavy chain constant region of anti-CD73 antibodies described hereinmay be of any isotype, e.g., IgG1, IgG2, IgG3 and IgG4, or combinationsthereof and/or modifications thereof. An anti-CD73 antibody may haveeffector function or may have reduced or no effector function. Incertain embodiments, anti-CD73 antibodies described herein comprise amodified heavy chain constant region that provides enhanced propertiesto the antibody. As shown in the Examples, anti-CD73 antibodies havingan IgG2 hinge and optionally an IgG2 CH1 domain, such as those havingthe variable regions of the 11F11 antibody, are better and fasterinternalized relative to antibodies having the same variable region butwith a non-IgG2 hinge or CH1, e.g., relative to antibodies having anIgG1 hinge or an IgG1 hinge and IgG1 CH1. For example an antibodycomprising the variable regions of the 11F11 antibody and comprising anIgG2 hinge and optionally an IgG2 CH1 and an IgG1 CH2 and IgG1 CH3domains, and whether with or without effector function, is moreefficiently internalized into cells upon binding to CD73 on the cellmembrane relative to the same antibody, but with an IgG1 hinge or anIgG1 hinge and IgG1 CH1 domain. As further shown herein, a CD73 antibodyhaving an IgG2 hinge and the remainder of the antibody of an IgG1isotype internalizes more efficiently than the same antibody wherein thehinge is of an IgG1 isotype. An antibody having, in addition to an IgG2hinge, an IgG2 CH1 domain internalizes even more efficiently than thesame antibody in which the CH1 domain is an IgG1 CH1 domain. As furthershown herein, anti-CD73 antibodies with an IgG2 hinge and optionallyIgG2 CH1 also form larger antibody/antigen complexes than antibodieshaving an IgG1 hinge or IgG1 hinge and IgG1 CH1. Increasedinternalization appears to correlate with increased antibody/antigencomplex size. As further described in the Examples, enhancedinternalization does not appear to be associated with a higher or loweraffinity of the antibody. Accordingly, provided herein are anti-CD73antibodies having a modified heavy chain constant region that mediatesantibody mediated CD73 internalization, and wherein the antibody withthe modified heavy chain constant region binds to CD73 with a similaraffinity as the same antibody, but with a different heavy chain constantregion.

In certain embodiments, a CD73 antibody comprises a modified heavy chainconstant region that comprises a hinge of the IgG2 isotype (an “IgG2hinge”) and a CH1, CH2 and CH3 domain. In certain embodiments, amodified heavy chain constant region comprises an IgG2 hinge and a CH1,CH2 and CH3 domain, wherein at least one of the CH1, CH2 and CH3 domainsis not of the IgG2 isotype. In certain embodiments, a modified heavychain constant region comprises a hinge of the IgG2 isotype, a CH1 ofthe IgG2 isotype, wherein at least one of the CH2 and CH3 domains is notof the IgG2 isotype. The IgG2 hinge may be a wildtype IgG2 hinge, e.g.,a wildtype human IgG2 hinge (e.g., having SEQ ID NO:136) or a variantthereof, provided that the IgG2 hinge retains the ability to confer tothe antibody an enhanced activity (e.g., increased internalization by acell; enhanced inhibition of enzymatic activity; increased antagonist orblocking activity; the ability to form large antibody/antigencross-linked complexes; increased ability to stimulate or enhance animmune response; and/or increased anti-proliferative or anti-tumoreffect) relative to that of the same antibody that comprises a non-IgG2hinge and optionally a non-IgG2 CH1 domain. In certain embodiments, anIgG2 hinge variant retains similar rigidity or stiffness to that of awildtype IgG2 hinge. The rigidity of a hinge or an antibody can bedetermined, e.g., by computer modeling, electron microscopy,spectroscopy such as Nuclear Magnetic Resonance (NMR), X-raycrystallography (B-factors), or Sedimentation Velocity Analyticalultracentrifugation (AUC) to measure or compare the radius of gyrationof antibodies comprising the hinge. A hinge or antibody may have similaror higher rigidity relative to another hinge if an antibody comprisingthe hinge has a value obtained from one of the tests described in theprevious sentence that differs from the value of the same antibody witha different hinge, e.g., an IgG1 hinge, in less than 5%, 10%, 25%, 50%,75%, or 100%. A person of skill in the art would be able to determinefrom the tests whether a hinge or an antibody has at least similarrigidity to that of another hinge or antibody, respectively, byinterpreting the results of these tests. An exemplary human IgG2 hingevariant is an IgG2 hinge that comprises a substitution of one or more ofthe four cysteine residues (i.e., C219, C220, C226 and C229) withanother amino acid. A cysteine may be replaced by a serine. An exemplaryIgG2 hinge is a human IgG2 hinge comprising a C219X mutation or a C220Xmutation, wherein X is any amino acid except serine. In a certainembodiments, an IgG2 hinge does not comprise both a C219X and a C220Xsubstitution. In certain embodiments, an IgG2 hinge comprises C219S orC220S, but not both C219S and C220S. Other IgG2 hinge variants that maybe used include human IgG2 hinges comprising a C220, C226 and/or C229substitution, e.g., a C220S, C226S or C229S mutation (which may becombined with a C219S mutation). An IgG2 hinge may also be an IgG2 hingein which a portion of the hinge is that of another isotype (i.e., it isa chimeric or hybrid hinge), provided that the rigidity of the chimerichinge is at least similar to that of a wildtype IgG2 hinge. For example,an IgG2 hinge may be an IgG2 hinge in which the lower hinge (as definedin Table 2) is of an IgG1 isotype, and is, e.g., a wildtype IgG1 lowerhinge.

A “hybrid” or “chimeric” hinge is referred to as being of a specificisotype if more than half of the consecutive amino acids of the hingeare from that isotype. For example, a hinge having an upper and middlehinge of IgG2 and the lower hinge of IgG1 is considered to be an IgG2hybrid hinge.

In certain embodiments, a CD73 antibody comprises a modified heavy chainconstant region that comprises an IgG2 hinge comprising one of thefollowing hinges:

ERKCCVECPPCPAPPVAG; (SEQ ID NO: 348) ERKSCVECPPCPAPPVAG;(SEQ ID NO: 349) ERKCSVECPPCPAPPVAG; (SEQ ID NO: 350)ERKXCVECPPCPAPPVAG; (SEQ ID NO: 351) ERKCXVECPPCPAPPVAG;(SEQ ID NO: 352) ERKCCVECPPCPAPPVAGX; (SEQ ID NO: 353)ERKSCVECPPCPAPPVAGX; (SEQ ID NO: 354) ERKCSVECPPCPAPPVAGX;(SEQ ID NO: 355) ERKXCVECPPCPAPPVAGX; (SEQ ID NO: 356)ERKCXVECPPCPAPPVAGX; (SEQ ID NO: 357) ERKCCVECPPCPAPELLGG;(SEQ ID NO: 358) ERKSCVECPPCPAPELLGG; (SEQ ID NO: 359)ERKCCSVECPPCPAPELLGG; (SEQ ID NO: 360) ERKXCVECPPCPAPELLGG;(SEQ ID NO: 361) ERKCXVECPPCPAPELLGG; (SEQ ID NO: 362)ERKCCVECPPCPAPELLG; (SEQ ID NO: 363) ERKSCVECPPCPAPELLG;(SEQ ID NO: 364) ERKCCSVECPPCPAPELLG; (SEQ ID NO: 365)ERKXCVECPPCPAPELLG; (SEQ ID NO: 366) ERKCXVECPPCPAPELLG;(SEQ ID NO: 367) ERKCCVECPPCPAP; (SEQ ID NO: 368) ERKSCVECPPCPAP;(SEQ ID NO: 369) ERKCSVECPPCPAP; (SEQ ID NO: 370) ERKXCVECPPCPAP;(SEQ ID NO: 371) or ERKCXVECPPCPAP, (SEQ ID NO: 372)

wherein X is any amino acid, except a cysteine,

or any of the above sequences, in which 1-5, 1-3, 1-2 or 1 amino acid isinserted between amino acid residues CVE and CPP. In certainembodiments, THT or GGG is inserted.

In certain embodiments, the hinge comprises SEQ ID NO: 348, 349, 350,351, or 352, wherein 1, 2, 3 or all 4 amino acids P233, V234, A235 andG237 (corresponding to the C-terminal 4 amino acids “PVAG” (SEQ ID NO:373) are deleted or substituted with another amino acid, e.g., the aminoacids of the C-terminus of the IgG1 hinge (ELLG (SEQ ID NO: 374) orELLGG (SEQ ID NO: 375). In certain embodiments, the hinge comprises SEQID NO: 348, 349, 350, 351, or 352, wherein V234, A235 and G237 aredeleted or substituted with another amino acid. In certain embodiments,the hinge comprises SEQ ID NO: 348, 349, 350, 351, or 352, wherein A235and G237 are deleted or substituted with another amino acid. In certainembodiments, the hinge comprises SEQ ID NO: 348, 349, 350, 351, or 352,wherein G237 is deleted or substituted with another amino acid. Incertain embodiments, the hinge comprises SEQ ID NO: 348, 349, 350, 351,or 352, wherein V234 and A235 are deleted or substituted with anotheramino acid. Substitution of PVAG (SEQ ID NO: 373) in an IgG2 with thecorresponding amino acids of an IgG1 hinge, i.e., (ELLG (SEQ ID NO: 374)or ELLGG (SEQ ID NO: 375)) to obtain a hybrid hinge, e.g., shown above,provides a hinge having the advantages of an IgG2 hinge and the effectorfunction of IgG1 hinges.

In certain embodiments, a modified heavy chain constant region comprisesa hinge that consists of or consists essentially of one of the sequencesshown above, e.g., any one of SEQ ID NOs: 348-372, and e.g., does notcomprise additional hinge amino acid residues.

In certain embodiments, 1 or 1-2 or 1-3 amino acids are inserted betweenthe hinge and CH2 domain, e.g., an additional glycine may be added.

In certain embodiments an anti-CD73 antibody comprises a modified heavychain constant region comprising an IgG1 or IgG2 constant region,wherein the hinge comprises a deletion of 1-10 amino acids. As shown inthe Examples, an IgG1 antibody lacking amino acid residues SCDKTHT(S219, C220, D221, K222, T223, H224 and T225; SEQ ID NO: 376) conferredantibody mediated CD73 internalization more effectively than the sameantibody having a wildtype IgG1 constant region. Similarly, in thecontext of an IgG2 antibody, an IgG2 antibody lacking amino acidresidues CCVE (C219, C220, V222, and E224; SEQ ID NO: 377) conferredantibody mediated CD73 internalization more effectively than the sameantibody having a wildtype IgG1 constant region. Accordingly, providedherein are modified heavy chain constant region in which the hingecomprises a deletion of 1, 2, 3, 4, 5, 6, or 7 amino acid residues,selected from residues 5219, C220, D221, K222, T223, H224 and T225 foran IgG1 antibody, and residues C219, C220, V222, and E224 for an IgG2antibody.

In certain embodiments, a modified heavy chain constant region comprisesa CH1 domain that is a wildtype CH1 domain of the IgG1 or IgG2 isotype(“IgG1 CH1 domain” or “IgG2 CH1 domain,” respectively). CH1 domains ofthe isotypes IgG3 and IgG4 (“IgG3 CH1 domain and “IgG2 CH1 domain,”respectively) may also be used. A CH1 domain may also be a variant of awildtype CH1 domain, e.g., a variant of a wildtype IgG1, IgG2, IgG3 orIgG4 CH1 domain. Exemplary variants of CH1 domains include A114C, T173Cand/or C131, e.g., C131S.

A CH1 domain, e.g., an IgG2 CH1 domain, may comprise the substitutionC131S, which substitution confers onto an IgG2 antibody or antibodyhaving an IgG2 CH1 and hinge the B form (or conformation).

In certain embodiments, a modified heavy chain constant region comprisesa CH1 domain that is of the IgG2 isotype. In certain embodiments, theCH1 domain is wildtype IgG2 CH1 domain, e.g., having the amino acidsequence: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV (SEQ ID NO: 378). In certainembodiments, the CH1 domain is a variant of SEQ ID NO: 378 and comprises1-10, 1-5, 1-2 or 1 amino acid substitutions or deletions relative toSEQ ID NO: 378. As further described in the Examples, it has been shownherein that an IgG2 CH1 domain or variants thereof confer enhanced oraltered internalization properties to anti-CD73 antibodies relative toIgG1 antibodies and even more enhanced or altered internalization whenthe antibodies also comprise an IgG2 hinge. In certain embodiments, IgG2CH1 variants do not comprise an amino acid substitution or deletion atone or more of the following amino acid residues: C131, R133, E137 and5138, which amino acid residues are shown in bold and underlined in SEQID NO: 378 shown above. For example, a modified heavy chain constantregion may comprise an IgG2 CH1 domain in which neither of R133, E137and 5138 are substituted with another amino acid or are deteled or inwhich neither of C131, R133, E137 and 5138 are substituted with anotheramino acid or are deteled. In certain embodiments, C131 is substitutedwith another amino acid, e.g., C131S, which substitution triggers theantibody to adopt conformation B. Both conformation A and conformation Bantibodies having modified heavy chain constant regions have been shownherein to have enhanced activities relative to the same antibody with anIgG1 constant region.

In certain embodiments, N192 and/or F193 (shown as italicized andunderlined residues in SEQ ID NO: 378 shown above) are substituted withanother amino acid, e.g., with the corresponding amino acids in IgG1,i.e., N192S and/or F193L.

In certain embodiments, one or more amino acid residues of an IgG2 CH1domain are substituted with the corresponding amino acid residues inIgG4. For example, N192 may be N192S; F193 may be F193L; C131 may beC131K; and/or T214 may be T214R.

An antibody may comprise a modified heavy chain constant regioncomprising an IgG2 CH1 domain or variant thereof and IgG2 hinge orvariant thereof. The hinge and CH1 domain may be a combination of anyIgG2 hinge and IgG2 CH1 domain described herein. In certain embodiments,the IgG2 CH1 and hinge comprise the following amino acid sequenceASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV

(SEQ ID NO: 379), or an amino acid sequence that differs therefrom in atmost 1-10 amino acids. The amino acid variants are as described for thehinge and CH1 domains above.

In certain embodiments, antibodies comprise at least an IgG2 hinge, andoptionally also an IgG2 CH1 domain or fragment or derivative of thehinge and/or CH1 domain and the antibody has adopted form (ofconformation) A (see, e.g., Allen et al. (2009) Biochemistry 48:3755).In certain embodiments, anti-CD73 antibodies comprise at least an IgG2hinge, and optionally also an IgG2 CH1 domain or fragment or derivativeof the hinge and/or CH1 domain and the antibody has adopted form B (see,e.g., Allen et al. (2009) Biochemistry 48:3755).

In certain embodiments, a modified heavy chain constant region comprisesa CH2 domain that is a wildtype CH2 domain of the IgG1, IgG2, IgG3 orIgG4 isotype (“IgG1 CH2 domain,” “IgG2 CH2 domain,” “IgG3 CH2 domain,”or “IgG4 CH2 domain,” respectively). A CH2 domain may also be a variantof a wildtype CH2 domain, e.g., a variant of a wildtype IgG1, IgG2, IgG3or IgG4 CH2 domain. Exemplary variants of CH2 domains include variantsthat modulate a biological activity of the Fc region of an antibody,such as ADCC or CDC or modulate the half-life of the antibody or itsstability. In one embodiment, the CH2 domain is a human IgG1 CH2 domainwith an A330S and P331S mutation, wherein the CH2 domain has reducedeffector function relative to the same CH2 mutation without themutations. A CH2 domain may have enhanced effector function. CH2 domainsmay comprise one or more of the following mutations: SE (S267E), SELF(S267E/L328F), SDIE (S239D/I332E), SEFF and GASDALIE(G236A/S239D/A330L/I332E) and/or one or more mutations at the followingamino acids: E233, G237, P238, H268, P271, L328 and A330. Othermutations are further set forth herein elsewhere.

In certain embodiments, a modified heavy chain constant region comprisesa CH3 domain that is a wildtype CH3 domain of the IgG1, IgG2, IgG3 orIgG4 isotype (“IgG1 CH3 domain,” “IgG2 CH3 domain,” “IgG3 CH3 domain,”or “IgG4 CH3 domain,” respectively. A CH3 domain may also be a variantof a wildtype CH3 domain, e.g., a variant of a wildtype IgG1, IgG2, IgG3or IgG4 CH3 domain. Exemplary variants of CH3 domains include variantsthat modulate a biological activity of the Fc region of an antibody,such as ADCC or CDC or modulate the half-life of the antibody or itsstability.

In certain embodiments, a modified heavy chain constant region comprisesa hinge of the IgG2 isotype and a CH1 region of the IgG2 isotype. TheIgG2 hinge and CH1 may be wild type IgG2 hinge and CH1 or variantsthereof, provided that they have the desired biological activity. Incertain embodiments, a modified heavy chain constant region comprises anIgG2 hinge comprising the C219S mutation, and an IgG2 CH1, which may bewild type or comprise at most 1-10, 1-5, 1-3, 1-2 or 1 amino acidsubstitution, deletion or addition. The modified heavy chain constantregion may further comprise a wild type or mutated CH2 and CH3 domains.For example, a CD73 antibody may comprise a heavy chain constant domaincomprising an IgG2 CH1 domain, an IgG2 hinge, which may comprise C219S,and an IgG1 CH2 and CH3 domain, wherein the CH2 and CH3 domain may beeffectorless, such as comprising mutations A330S and P331S.

Generally, variants of the CH1, hinge, CH2 or CH3 domains may comprise1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations, and/or at most 10, 9,8, 7, 6, 5, 4, 3, 2 or 1 mutation, or 1-10 or 1-5 mutations, or comprisean amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical to that of the corresponding wildtypedomain (CH1, hinge, CH2, or CH3 domain, respectively), provided that theheavy chain constant region comprising the specific variant retains thenecessary biological activity.

Table 5 sets forth exemplary human heavy chain constant regionscomprising a human CH1, hinge, CH2 and/or CH3 domains, wherein eachdomain is either a wildtype domain or a variant thereof that providesthe desired biological activity to the heavy chain constant region. Anunfilled cell in Table 5 indicates that the domain is present or not,and if present can be of any isotype, e.g., IgG1, IgG2, IgG3 or IgG4.For example, an antibody comprising the heavy chain constant region 1 inTable 5 is an antibody that comprises a heavy chain constant regioncomprising at least an IgG2 hinge, and which may also comprise a CH1,CH2 and/or CH3 domain, and if present, which CH1, CH2 and/or CH3 domainis of an IgG1, IgG2, IgG3 or IgG4 isotype. As another example forunderstanding Table 5, an antibody comprising a heavy chain constantregion 8 is an antibody comprising a heavy chain constant regioncomprising an IgG1 CH1 domain, and IgG2 hinge, an IgG1 CH2 domain, andwhich may or may not also comprise a CH3 domain, which is present, maybe of an IgG1, IgG2, IgG3 or IgG4 isotype.

TABLE 5 MHCCR* CH1 Hinge CH2 CH3 1 IgG2 2 IgG1 IgG2 3 IgG2 IgG2 4 IgG2IgG1 5 IgG2 IgG2 6 IgG2 IgG1 7 IgG2 IgG2 8 IgG1 IgG2 IgG1 9 IgG1 IgG2IgG2 10 IgG2 IgG2 IgG1 11 IgG2 IgG2 IgG2 12 IgG1 IgG2 IgG1 13 IgG1 IgG2IgG2 14 IgG2 IgG2 IgG1 15 IgG2 IgG2 IgG2 16 IgG2 IgG1 IgG1 17 IgG2 IgG1IgG2 18 IgG2 IgG2 IgG1 19 IgG2 IgG2 IgG2 20 IgG1 IgG2 IgG1 IgG1 21 IgG1IgG2 IgG1 IgG2 22 IgG1 IgG2 IgG2 IgG1 23 IgG1 IgG2 IgG2 IgG2 24 IgG2IgG2 IgG1 IgG1 25 IgG2 IgG2 IgG1 IgG2 26 IgG2 IgG2 IgG2 IgG1 27 IgG2IgG2 IgG2 IgG2 *Modified heavy chain constant region

In certain embodiments, an antibody comprising a heavy chain constantregion shown in Table 5 has an enhanced biological activity relative tothe same antibody comprising a heavy chain constant region that does notcomprise that specific heavy chain constant region or relative to thesame antibody that comprises an IgG1 constant region.

In certain embodiments, a method for improving the biological activityof a CD73 antibody that comprises a non-IgG2 hinge and/or non-IgG2 CH1domain comprises providing an anti-CD73 antibody that comprises anon-IgG2 hinge and/or a non-IgG2 CH1 domain, and replacing the non-IgG2hinge and the non-IgG2 CH1 domain with an IgG2 hinge and an IgG2 CH1domain, respectively. A method for improving the biological activity ofa CD73 antibody that does not comprise a modified heavy chain constantregion, may comprise providing an anti-CD73 antibody that does notcomprise a modified heavy chain constant region, and replacing its heavychain constant region with a modified heavy chain constant region.

Exemplary modified heavy chain constant regions that may be linked toanti-CD73 variable regions, e.g., those described herein, are providedin Table 6, which sets forth the identity of each of the domains.

TABLE 6 Modified heavy SEQ ID NO chain constant of whole region CH1Hinge CH2 CH3 MHCCR IgG1-IgG2- IgG1 wildtype IgG2/IgG1 IgG1 wildtypeIgG1 wildtype SEQ ID IgG1f SEQ ID NO: 98 SEQ ID NO: 178 SEQ ID NO: 137SEQ ID NO: 138 NO: 180 IgG1-IgG2- IgG1 wildtype IgG2 wildtype IgG1wildtype IgG1 wildtype SEQ ID IgG1f2 SEQ ID NO: 98 SEQ ID NO: 136 SEQ IDNO: 137 SEQ ID NO: 138 NO: 162 IgG1-IgG2CS- IgG1 wildtype IgG2C219S/IgG1IgG1 wildtype IgG1 wildtype SEQ ID IgG1f SEQ ID NO: 98 SEQ ID NO: 179SEQ ID NO: 137 SEQ ID NO: 138 NO: 181 IgG1-IgG2CS- IgG1 wildtype IgG2C219S IgG1 wildtype IgG1 wildtype SEQ ID IgG1f2 SEQ ID NO: 98 SEQ ID NO:123 SEQ ID NO: 137 SEQ ID NO: 138 NO: 163 IgG2-IgG1f IgG2 wildtypeIgG2/IgG1 IgG1 wildtype IgG1 wildtype SEQ ID SEQ ID NO: 124 SEQ ID NO:178 SEQ ID NO: 137 SEQ ID NO: 138 NO: 182 IgG2-IgG1f2 IgG2 wildtype IgG2wildtype IgG1 wildtype IgG1 wildtype SEQ ID SEQ ID NO: 124 SEQ ID NO:136 SEQ ID NO: 137 SEQ ID NO: 138 NO: 164 IgG2CS-IgG1f IgG2 wildtypeIgG2C219S/IgG1 IgG1 wildtype IgG1 wildtype SEQ ID SEQ ID NO: 124 SEQ IDNO: 179 SEQ ID NO: 137 SEQ ID NO: 138 NO: 183 IgG2CS-IgG1f2 IgG2wildtype IgG2 C219S IgG1 wildtype IgG1 wildtype SEQ ID SEQ ID NO: 124SEQ ID NO: 123 SEQ ID NO: 137 SEQ ID NO: 138 NO: 165 IgG1-IgG2- IgG1wildtype IgG2 wildtype IgG1 IgG1 wildtype SEQ ID IgG1.1f SEQ ID NO: 98SEQ ID NO: 136 A330S/P331S SEQ ID NO: 138 NO: 166 SEQ ID NO: 125IgG1-IgG2CS- IgG1 wildtype IgG2 C219S IgG1 IgG1 wildtype SEQ ID IgG1.1fSEQ ID NO: 98 SEQ ID NO: 123 A330S/P331S SEQ ID NO: 138 NO: 167 SEQ IDNO: 125 IgG2-IgG1.1f IgG2 wildtype IgG2 wildtype IgG1 IgG1 wildtype SEQID SEQ ID NO: 124 SEQ ID NO: 136 A330S/P331S SEQ ID NO: 138 NO: 168 SEQID NO: 125 IgG2CS-IgG1.1f IgG2 wildtype IgG2 C219S IgG1 IgG1 wildtypeSEQ ID SEQ ID NO: 124 SEQ ID NO: 123 A330S/P331S SEQ ID NO: 138 NO: 169SEQ ID NO: 125

In certain embodiments, an antibody comprises a modified heavy chainconstant region comprising an IgG2 hinge comprising SEQ ID NO: 123, 136,178, 179, or 348-372 or a variant thereof, such as an IgG2 hingecomprising an amino acid sequence that (i) differs from SEQ ID NO: 123,136, 178, 179, or 348-372 in 1, 2, 3, 4 or 5 amino acids substitutions,additions or deletions; (ii) differs from SEQ ID NO: 123, 136, 178, 179,or 348-372 in at most 5, 4, 3, 2, or 1 amino acids substitutions,additions or deletions; (iii) differs from SEQ ID NO: 123, 136, 178,179, or 348-372 in 1-5, 1-3, 1-2, 2-5 or 3-5 amino acids substitutions,additions or deletions and/or (iv) comprises an amino acid sequence thatis at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%identical to SEQ ID NO: 123, 136, 178, 179, or 348-372, wherein in anyof (i)-(iv), an amino acid substitution may be a conservative amino acidsubstitution or a non-conservative amino acid substitution; and whereinthe modified heavy chain constant region has an enhanced biologicalactivity relative to that of another heavy chain constant region, e.g.,a heavy chain constant region that comprises a non-IgG2 hinge orrelative to the same modified heavy chain constant region that comprisesa non-IgG2 hinge. For example, the hinge may be wildtype, or comprise aC219S, C220S or C219S and C220S substitutions.

In certain embodiments, an antibody comprises a modified heavy chainconstant region comprising an IgG1 CH1 domain comprising SEQ ID NO: 98or an IgG2 CH1 domain comprising SEQ ID NO: 124, or a variant of SEQ IDNO: 98 or 124, which variant (i) differs from SEQ ID NO: 98 or 124 in 1,2, 3, 4 or 5 amino acids substitutions, additions or deletions; (ii)differs from SEQ ID NO: 98 or 124 in at most 5, 4, 3, 2, or 1 aminoacids substitutions, additions or deletions; (iii) differs from SEQ IDNO: 98 or 124 in 1-5, 1-3, 1-2, 2-5 or 3-5 amino acids substitutions,additions or deletions and/or (iv) comprises an amino acid sequence thatis at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%identical to SEQ ID NO: 98 or 124, wherein in any of (i)-(iv), an aminoacid substitution may be a conservative amino acid substitution or anon-conservative amino acid substitution; and wherein the modified heavychain constant region has an enhanced biological activity relative tothat of another heavy chain constant region, e.g., a heavy chainconstant region that comprises a non-IgG2 hinge or non-IgG2 hinge andCH1 domain or relative to the same modified heavy chain constant regionthat comprises a non-IgG2 hinge or non-IgG2 hinge and CH1 domain. AnIgG2 CH1 domain may comprise C131S or other mutations that causes anIgG2 hinge and CH1 containing antibody to adopt either an A or a B form.

In certain embodiments, an antibody comprises a modified heavy chainconstant region comprising an IgG1 CH2 domain comprising SEQ ID NO: 137or 125, or a variant of SEQ ID NO: 137 or 125, which variant (i) differsfrom SEQ ID NO: 137 or 125 in 1, 2, 3, 4 or 5 amino acids substitutions,additions or deletions; (ii) differs from SEQ ID NO: 137 or 125 in atmost 5, 4, 3, 2, or 1 amino acids substitutions, additions or deletions;(iii) differs from SEQ ID NO: 137 or 125 in 1-5, 1-3, 1-2, 2-5 or 3-5amino acids substitutions, additions or deletions and/or (iv) comprisesan amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%,96%, 97%, 98% or 99% identical to SEQ ID NO: 137 or 125, wherein in anyof (i)-(iv), an amino acid substitution may be a conservative amino acidsubstitution or a non-conservative amino acid substitution; and whereinthe modified heavy chain constant region has an enhanced biologicalactivity relative to that of another heavy chain constant region, e.g.,a heavy chain constant region that comprises a non-IgG2 hinge orrelative to the same modified heavy chain constant region that comprisesa non-IgG2 hinge.

In certain embodiments, an antibody comprises a modified heavy chainconstant region comprising an IgG1 CH3 domain comprising SEQ ID NO: 138,or a variant of SEQ ID NO: 138, which variant (i) differs from SEQ IDNO: 138 in 1, 2, 3, 4 or 5 amino acids substitutions, additions ordeletions; (ii) differs from SEQ ID NO: 138 in at most 5, 4, 3, 2, or 1amino acids substitutions, additions or deletions; (iii) differs fromSEQ ID NO: 138 in 1-5, 1-3, 1-2, 2-5 or 3-5 amino acids substitutions,additions or deletions and/or (iv) comprises an amino acid sequence thatis at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%identical to SEQ ID NO: 138, wherein in any of (i)-(iv), an amino acidsubstitution may be a conservative amino acid substitution or anon-conservative amino acid substitution; and wherein the modified heavychain constant region has an enhanced biological activity relative tothat of another heavy chain constant region, e.g., a heavy chainconstant region that comprises a non-IgG2 hinge or relative to the samemodified heavy chain constant region that comprises a non-IgG2 hinge.

Modified heavy chain constant regions may also comprise a combination ofthe CH1, hinge, CH2 and CH3 domains described above.

In certain embodiments, a CD73 antibody comprises a modified heavy chainconstant region comprising any one of SEQ ID NOs: 162-169, 180-183,267-282, and 300-347, or a variant of any one of SEQ ID NOs: 162-169,180-183, 267-282, and 300-347, which variant (i) differs from SEQ IDNOs: 162-169, 180-183, 267-282, and 300-347 in 1, 2, 3, 4, 5, 6, 7, 8,9, 10 or more amino acids substitutions, additions or deletions; (ii)differs from SEQ ID NOs: 162-169, 180-183, 267-282, and 300-347 in atmost 10, 9, 8, 7, 6,5, 4, 3, 2, or 1 amino acids substitutions,additions or deletions; (iii) differs from SEQ ID NOs: 162-169, 180-183,267-282, and 300-347 in 1-5, 1-3, 1-2, 2-5, 3-5, 1-10, or 5-10 aminoacids substitutions, additions or deletions and/or (iv) comprises anamino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% identical to any one of SEQ ID NOs: 162-169, 180-183,267-282, and 300-347, wherein in any of (i)-(iv), an amino acidsubstitution may be a conservative amino acid substitution or anon-conservative amino acid substitution; and wherein the modified heavychain constant region has an enhanced biological activity relative tothat of another heavy chain constant region, e.g., a heavy chainconstant region that comprises a non-IgG2 hinge or non-IgG2 CH1 domainor relative to the same modified heavy chain constant region thatcomprises a non-IgG2 hinge and/or a non-IgG2 CH1 domain.

Modified heavy chain constant regions may have (i) similar, reduced orincreased effector function (e.g., binding to an FcγR, e.g., FcγRIIB)relative to a wildtype heavy chain constant region and or (ii) similar,reduced or increased half-life (or binding to the FcRn receptor)relative to a wildtype heavy chain constant region.

The VH domain of an anti-CD73 antibody described herein may be linked toa heavy chain constant region described herein. For example, FIG. 18shows the amino acid sequence of antibody CD73.4 linked to the heavychain constant region IgG2CS-IgG1.1f (SEQ ID NO:133 or 169). Alsoencompassed herein are antibodies comprising a heavy chain comprising anamino acid sequence that differs from that of CD73.4-IgG2CS-IgG1.1f (SEQID NO:133 or 189) in at most 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4,1-3, 1-2 or 1 amino acid (by substitution, addition or deletion) and/orthat are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical tothe amino acid sequence of the heavy chain of CD73.4-IgG2CS-IgG1.1f (SEQID NO:133 or 189). For example, encompassed herein are antibodiescomprising the heavy chain of CD73.4-IgG2CS-IgG1.1f (SEQ ID NO: 133 or189), and wherein the C-terminal K or GK or PGK are deleted or arepresent. Other variants of CD73.4-IgG2CS-IgG1.1f (SEQ ID NO:133 or 189)include those having a heavy chain that is of a different allotype, andwherein, e.g., amino acids 356 and 358 are D and L, respectively.Variants include those having an additional cysteine mutated in the IgG2hinge, e.g., C220 (or have C220S instead of C219S), and those that donot have the mutations A330S and/or P331S. Variants ofCD73.4-IgG2CS-IgG1.1f (SEQ ID NO:133 or 189) preferably have at leastsimilar biochemical properties and/or biological activities, e.g.,efficiency of internalization, inhibition of CD73 enzymatic activity,affinity for human CD73, and binding to the same or similar epitope,relative to CD73.4-IgG2CS-IgG1.1f (SEQ ID NO:133 or 189).

In certain embodiments, the anti-CD73 antibodies, or antigen bindingportions thereof, comprise any one of the constant regions describedherein, e.g., constant regions comprising the amino acid sequences setforth in SEQ ID NOs: 126, 127, 129, 130, 162-169, 180-183, 267-282, and300-347.

A light chain of an anti-CD73 antibody may comprise a light chainconstant region comprising SEQ ID NO: 131, or a variant of SEQ ID NO:131, which variant (i) differs from SEQ ID NO: 131 in 1, 2, 3, 4, 5, 6,7, 8, 9, 10 or more amino acids substitutions, additions or deletions;(ii) differs from SEQ ID NO: 131 in at most 10, 9, 8, 7, 6,5, 4, 3, 2,or 1 amino acids substitutions, additions or deletions; (iii) differsfrom SEQ ID NO: 131 in 1-5, 1-3, 1-2, 2-5, 3-5, 1-10, or 5-10 aminoacids substitutions, additions or deletions and/or (iv) comprises anamino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% identical to SEQ ID NO: 131, wherein in any of (i)-(iv),an amino acid substitution may be a conservative amino acid substitutionor a non-conservative amino acid substitution. An exemplary CL mutationincludes C124S.

Heavy and light chains comprising an amino acid sequence that is atleast 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75% or 70% identical toany of the heavy or light chains set forth in Table 35, as detailedherein (or their variable regions), may be used for forming anti-humanCD73 antibodies having the desired characteristics, e.g., those furtherdescribed herein. Exemplary variants are those comprising an allotypicvariation, e.g., in the constant domain. Heavy and light chainscomprising an amino acid sequence that differs in at most 1-30, 1-25,1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2 or 1 amino acid (by substitution,addition or deletion) from any of the heavy or light chains set forth inTable 35, as described herein (or their variable regions), may be usedfor forming anti-human CD73 antibodies having the desiredcharacteristics, e.g., those further described herein.

In various embodiments, the antibodies described above exhibit one ormore, two or more, three or more, four or more, five or more, six ormore, seven or more, eight or more, nine or more, ten, or all of thefunctional properties listed in Table 3.

Such antibodies include, for example, human antibodies, humanizedantibodies, or chimeric antibodies.

In one embodiment, the anti-CD73 antibodies described herein bind toboth glycosylated (e.g., N-linked or O-linked glycosylation) andunglycosylated human CD73. Certain anti-CD73 antibodies may bind toglycosylated, but not unglycosylated CD73 or to unglycosylated but notglycosylated CD73.

In one embodiment, the anti-CD73 antibodies described herein bind to aconformational epitope.

In one embodiment, the anti-CD73 antibodies described herein bind toamino acid residues within the following region of human CD73:

FTKVQQIRRAEPNVLLLDA (SEQ ID NO: 96) and

corresponding to amino acid residues 65-83 of human CD73 (SEQ ID NO: 1or 2), as determined by, e.g., HDX-MS.

In one embodiment, the anti-CD73 antibodies described herein bind to allor a portion of the following amino acid residues in human CD73:FTKVQQIRRAEPNVLLLDA (SEQ ID NO: 96), which corresponds to amino acidresidues 65-83 of human CD73 (SEQ ID NO: 1 or 2), as determined by,e.g., HDX-MS.

In one embodiment, the anti-CD73 antibody described herein binds toamino acid residues within the following region of human CD73:

-   -   LYLPYKVLPVGDEVVG (SEQ ID NO: 97),        corresponding to amino acid residues 157-172 of human CD73 (SEQ        ID NO: 1 or 2), as determined by, e.g., HDX-MS.

In one embodiment, the anti-CD73 antibody described herein binds to allor a portion of the following amino acid residues within human CD73:LYLPYKVLPVGDEVVG (SEQ ID NO: 97), which corresponds to amino acidresidues 157-172 of human CD73 (SEQ ID NO: 1 or 2), as determined by,e.g., HDX-MS.

In one embodiment, the anti-CD73 antibody described herein binds todiscontinuous amino acid residues within the following regions of humanCD73 (SEQ ID NO: 1 or 2):

FTKVQQIRRAEPNVLLLDA (SEQ ID NO: 96) and LYLPYKVLPVGDEVVG.(SEQ ID NO: 97)

In one embodiment, the anti-CD73 antibody described herein binds to allor a portion of the discontinuous amino acid residues within thefollowing regions of human CD73 (SEQ ID NO: 1 or 2): FTKVQQIRRAEPNVLLLDA(SEQ ID NO: 96) and LYLPYKVLPVGDEVVG (SEQ ID NO: 97), which correspondto amino acid residues 65-83 and 157-172 of human CD73 (SEQ ID NO: 1 or2), as determined by, e.g., HDX-MS.

In certain embodiments, anti-CD73 antibodies have interactions withhuman CD73 that correspond to those shown in Table 30, as determined byX-ray crystallography. An antibody may share at least 50%, 60%, 70%,80%, 90%, 95% or 99% of the interactions with human CD73 that are shownin Table 30.

III. Antibodies Having Particular Germline Sequences

In certain embodiments, an anti-CD73 antibody comprises a heavy chainvariable region from a particular germline heavy chain immunoglobulingene and/or a light chain variable region from a particular germlinelight chain immunoglobulin gene.

As demonstrated herein, human antibodies specific for CD73 have beenprepared that comprise a heavy chain variable region that is the productof or derived from a human germline VH 3-33 gene, VH 3-10 gene, VH 3-15gene, VH 3-16, JH6b gene, VH 6-19 gene, VH 4-34 gene, and/or JH3b gene.Accordingly, provided herein are isolated monoclonal antibodies specificfor human CD73, or antigen-binding portions thereof, comprising a heavychain variable region that is the product of or derived from a human VHgermline gene selected from the group consisting of: VH 3-33, VH 3-10,VH 3-15, VH 3-16, VH 6-19, and VH 4-34.

Human antibodies specific for CD73 have been prepared that comprise alight chain variable region that is the product of or derived from ahuman germline VK L6 gene, VK L18 gene, VK L15 gene, VK L20 gene, VK A27gene, JK5 gene, JK4 gene, JK2 gene, and JK1 gene. Accordingly, providedherein are isolated monoclonal antibodies specific for human CD73, orantigen-binding portions thereof, comprising a light chain variableregion that is the product of or derived from a human VK germline geneselected from the group consisting of: VK L6, VK L18, VK L15, VK L20,and VK A27.

Preferred antibodies described herein are those comprising a heavy chainvariable region that is the product of or derived from one of theabove-listed human germline VH genes and also comprising a light chainvariable region that is the product of or derived from one of theabove-listed human germline VK genes.

As used herein, a human antibody comprises heavy or light chain variableregions that are “the product of” or “derived from” a particulargermline sequence if the variable regions of the antibody are obtainedfrom a system that uses human germline immunoglobulin genes. Suchsystems include immunizing a transgenic mouse carrying humanimmunoglobulin genes with the antigen of interest or screening a humanimmunoglobulin gene library displayed on phage with the antigen ofinterest. A human antibody that is “the product of” or “derived from” ahuman germline immunoglobulin sequence can be identified as such bycomparing the amino acid sequence of the human antibody to the aminoacid sequences of human germline immunoglobulins and selecting the humangermline immunoglobulin sequence that is closest in sequence (i.e.,greatest % identity) to the sequence of the human antibody. A humanantibody that is “the product of” or “derived from” a particular humangermline immunoglobulin sequence may contain amino acid differences ascompared to the germline sequence, due to, for example,naturally-occurring somatic mutations or intentional introduction ofsite-directed mutation. However, a selected human antibody typically isat least 90% identical in amino acids sequence to an amino acid sequenceencoded by a human germline immunoglobulin gene and contains amino acidresidues that identify the human antibody as being human when comparedto the germline immunoglobulin amino acid sequences of other species(e.g., murine germline sequences). In certain cases, a human antibodymay be at least 95%, or even at least 96%, 97%, 98%, or 99% identical inamino acid sequence to the amino acid sequence encoded by the germlineimmunoglobulin gene. Typically, a human antibody derived from aparticular human germline sequence will display no more than 10 aminoacid differences from the amino acid sequence encoded by the humangermline immunoglobulin gene. In certain cases, the human antibody maydisplay no more than 5, or even no more than 4, 3, 2, or 1 amino aciddifference from the amino acid sequence encoded by the germlineimmunoglobulin gene.

IV. Homologous Antibodies

Encompassed herein are antibodies having heavy and light chain variableregions comprising amino acid sequences that are homologous to the aminoacid sequences of the preferred antibodies described herein, and whereinthe antibodies retain the desired functional properties of the anti-CD73antibodies described herein.

For example, an isolated anti-CD73 antibody, or antigen binding portionthereof, may comprise a heavy chain variable region and a light chainvariable region, wherein:

(a) the heavy chain variable region comprises an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical toan amino acid sequence selected from the group consisting of SEQ ID NOs:4, 16, 32, 40, 52, 60, 68, 80, 88, 135, and 170-177, or comprises 1, 2,3, 4, 5, 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25, or 1-50 amino acidchanges (i.e., amino acid substitutions, additions or deletions)relative to an amino acid sequence selected from the group consisting ofSEQ ID NOs: 4, 16, 32, 40, 52, 60, 68, 80, 88, 135, and 170-177,respectively;

(b) the light chain variable region comprises an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical toan amino acid sequence selected from the group consisting of SEQ ID NOs:8, 12, 20, 24, 28, 36, 44, 48, 56, 64, 72, 76, 84, 92, and 138, orcomprises 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25, or1-50 amino acid changes (i.e., amino acid substitutions, additions ordeletions) relative to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 8, 12, 20, 24, 28, 36, 44, 48, 56, 64, 72, 76,84, 92, and 238, respectively;

(c) the antibody specifically binds to CD73, and

(d) the antibody exhibits 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of thefunctional properties listed in Table 3.

In certain embodiments, the anti-CD73 antibodies comprise heavy andlight chain variable regions with the percent identities and/or aminoacid changes and functions discussed above (i.e., (a)-(d)), wherein theCDR3 of the heavy chain variable region comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 7, 19, 35, 43, 55, 63,71, 83, and 91, and optionally the CDR1 of the heavy chain variableregion comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 5, 17, 33, 41, 53, 61, 69, 81, and 89, andoptionally the CDR2 of the heavy chain variable region comprises anamino acid sequence selected from the group consisting of SEQ ID NOs: 6,18, 34, 42, 54, 62, 70, 82, and 90.

In certain embodiments, the anti-CD73 antibodies comprise heavy andlight chain variable regions with the percent identities and/or aminoacid changes and functions discussed above (i.e., (a)-(d)), wherein theCDR3 of the light chain variable region comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 11, 15, 23, 27, 31,39, 47, 51, 59, 67, 75, 79, 87, 95, and 241, and optionally the CDR1 ofthe light chain variable region comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 9, 13, 21, 25, 29, 37,45, 49, 57, 65, 73, 77, 85, 93, and 239, and optionally the CDR2 of thelight chain variable region comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 10, 14, 22, 26, 30, 38, 46, 50,58, 66, 74, 78, 86, 94, and 240.

In certain embodiments, the anti-CD73 antibodies comprise heavy andlight chain variable regions with the percent identities and/or aminoacid changes and functions discussed above (i.e., (a)-(d)), wherein theCDR3 of the heavy chain variable region comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 7, 19, 35, 43, 55, 63,71, 83, and 91, and optionally the CDR1 of the heavy chain variableregion comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 5, 17, 33, 41, 53, 61, 69, 81, and 89, andoptionally the CDR2 of the heavy chain variable region comprises anamino acid sequence selected from the group consisting of SEQ ID NOs: 6,18, 34, 42, 54, 62, 70, 82, and 90, and wherein the CDR3 of the lightchain variable region comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 11, 15, 23, 27, 31, 39, 47, 51, 59, 67,75, 79, 87, 95, and 241, and optionally the CDR1 of the light chainvariable region comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 9, 13, 21, 25, 29, 37, 45, 49, 57, 65, 73, 77,85, 93, and 239, and optionally the CDR2 of the light chain variableregion comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 10, 14, 22, 26, 30, 38, 46, 50, 58, 66, 74,78, 86, 94, and 240.

In various embodiments, the antibody can be, for example, a humanantibody, a humanized antibody or a chimeric antibody.

An isolated anti-CD73 antibody, or antigen binding portion thereof, maycomprise a heavy chain and a light chain, wherein:

(a) the heavy chain comprises an amino acid sequence that is at least80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to an amino acidsequence selected from the group consisting of SEQ ID NOs: 100, 103,107, 109, 112, 114, 116, 119, 121, 133, 184-210 or comprises 1, 2, 3, 4,5, 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25, or 1-50 amino acidchanges (i.e., amino acid substitutions, additions or deletions)relative to an amino acid sequence selected from the group consisting ofSEQ ID NOs: 100, 103, 107, 109, 112, 114, 116, 119, 121, 133, and184-210, respectively;

(b) the light chain comprises an amino acid sequence that is at least80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to an amino acidsequence selected from the group consisting of SEQ ID NOs: 101, 102,104, 105, 106, 108, 110, 111, 113, 115, 117, 118, 120 and 122 orcomprises 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25, or1-50 amino acid changes (i.e., amino acid substitutions, additions ordeletions) relative to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 101, 102, 104, 105, 106, 108, 110, 111, 113,115, 117, 118, 120 and 122, respectively;

(c) the antibody specifically binds to CD73, and

(d) the antibody exhibits 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of thefunctional properties listed in Table 3.

In certain embodiments, the anti-CD73 antibodies comprise heavy andlight chains with the percent identities and/or amino acid changes andfunctions discussed above (i.e., (a)-(d)), wherein the CDR3 of the heavychain variable region comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 7, 19, 35, 43, 55, 63, 71, 83, and 91,and optionally the CDR1 of the heavy chain variable region comprises anamino acid sequence selected from the group consisting of SEQ ID NOs: 5,17, 33, 41, 53, 61, 69, 81, and 89, and optionally the CDR2 of the heavychain variable region comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 6, 18, 34, 42, 54, 62, 70, 82, and 90.

In certain embodiments, the anti-CD73 antibodies comprise heavy andlight chains with the percent identities and/or amino acid changes andfunctions discussed above (i.e., (a)-(d)), wherein the CDR3 of the lightchain variable region comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 11, 15, 23, 27, 31, 39, 47, 51, 59, 67,75, 79, 87, 95, and 241, and optionally the CDR1 of the light chainvariable region comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 9, 13, 21, 25, 29, 37, 45, 49, 57, 65, 73, 77,85, 93, and 239, and optionally the CDR2 of the light chain variableregion comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 10, 14, 22, 26, 30, 38, 46, 50, 58, 66, 74,78, 86, 94, and 240.

In certain embodiments, the anti-CD73 antibodies comprise heavy andlight chains with the percent identities and/or amino acid changes andfunctions discussed above (i.e., (a)-(d)), wherein the CDR3 of the heavychain variable region comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 7, 19, 35, 43, 55, 63, 71, 83, and 91,and optionally the CDR1 of the heavy chain variable region comprises anamino acid sequence selected from the group consisting of SEQ ID NOs: 5,17, 33, 41, 53, 61, 69, 81, and 89, and optionally the CDR2 of the heavychain variable region comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 6, 18, 34, 42, 54, 62, 70, 82, and 90,and wherein the CDR3 of the light chain variable region comprises anamino acid sequence selected from the group consisting of SEQ ID NOs:11, 15, 23, 27, 31, 39, 47, 51, 59, 67, 75, 79, 87, 95, and 241, andoptionally the CDR1 of the light chain variable region comprises anamino acid sequence selected from the group consisting of SEQ ID NOs: 9,13, 21, 25, 29, 37, 45, 49, 57, 65, 73, 77, 85, 93, and 239, andoptionally the CDR2 of the light chain variable region comprises anamino acid sequence selected from the group consisting of SEQ ID NOs:10, 14, 22, 26, 30, 38, 46, 50, 58, 66, 74, 78, 86, 94, and 240.

Also provided are anti-CD73 antibodies comprising a VHCDR1, VHCDR2,VHCDR3, VLCDR1, VLCDR2, and/or VLCDR3 that differs from thecorresponding CDRs of CD73.4-1, CD73.4-2, CD73.3, 11F11-1, 11F11-2,4C3-1, 4C3-2, 4C3-3, 4D4, 10D2-1, 10D2-2, 11A6, 24H2, 5F8-1, 5F8-2, 6E11and/or 7A11, in 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, or 1-5 amino acid changes(i.e., amino acid substitutions, additions or deletions). In certainembodiments, an anti-CD73 antibody comprises 1-5 amino acid changes ineach of 1, 2, 3, 4, 5 or 6 of the CDRs relative to the correspondingsequences in CD73.4-1, CD73.4-2, CD73.3, 11F11-1, 11F11-2, 11F11, 4C3-1,4C3-2, 4C3-3, 4D4, 10D2-1, 10D2-2, 11A6, 24H2, 5F8-1, 5F8-2, 6E11 and/or7A11. In certain embodiments, an anti-CD73 antibody comprises at totalof 1-5 amino acid changes across all CDRs relative to the CDRs inCD73.4-1, CD73.4-2, CD73.3, 11F11-1, 11F11-2, 4C3-1, 4C3-2, 4C3-3, 4D4,10D2-1, 10D2-2, 11A6, 24H2, 5F8-1, 5F8-2, 6E11 and/or 7A11.

In certain embodiments, an anti-CD73 antibody comprises VH and VL CDRsconsisting of those of CD73.4-1 or CD73.4-2, wherein one or more of theamino acids in one or more CDRs are those of one of the other anti-CD73antibodies disclosed herein.

Mutations (e.g., substitutions, additions, deletions) that can be madein the variable region sequences of the anti-CD73 antibodies can bedetermined based on the following: (i) the mutations that wereintroduced into the antibodies, as described in the Examples; and (ii)the comparison of the amino acid residues at each position in thevariable domains of the anti-CD73 antibodies described herein (see Table35 and FIG. 35): a different amino acid at a certain position inanti-CD73 antibodies may indicate that the amino acid residue at thisposition may be changed to another amino acid residue withoutsignificantly affecting the activities of the antibody; whereas if thesame amino acid residue is found in the same position in several or allanti-CD73 antibodies, this may indicate that this particular amino acidshould be preserved and not changed to another residue. Exemplaryembodiments are provided below.

In certain embodiments, a framework substitution can be introduced atposition 25 ( . . . RLSCATSGFTF . . . in 11F11) of the heavy chainvariable region (e.g., a conservative substitution, e.g., to S or A) ofthe anti-CD73 antibodies described herein. For example, if the aminoacid at this position is T, a substitution to A or S can be introduced;if the amino acid at this position is A, a substitution to S or T can beintroduced; and if the amino acid at this position is S, a substitutionto T or A can be introduced. Antibodies 24H2, 4D4, 10D2, 6E11, 7A11,11A6, and 4C3 have an A at this position, 11F11 has a T at thisposition, and 73.5, 73.7, and 73.9 have an S at this position.

Similarly, in certain embodiments, a framework substitution can beintroduced at amino acid position 94 ( . . . AEDTAVYYCAR . . . in 11F11)of the heavy chain variable region (e.g., V to L or L to V). Forexample, antibodies 11F11, 73.3-73.10, 24H2, 4D4, 5F8, and 10D2 have a Vat this position, and 6E11, 7A11, 11A6, and 4C3 have an L at thisposition.

In certain embodiments, amino acid substitutions can be made to theheavy chain variable region CDR2 of the anti-CD73 antibodies disclosedherein. For example, the amino acid at position 52 ( . . . WVAVILYDGSN .. . in 11F11) can be substituted with W, or if the amino acid at thisposition is W, then the amino acid can be substituted with L (antibodies11F11 and 73.4-73.7 have an L at this position, and antibodies73.8-73.10, 24H2, and 4D4 have a W at this position).

Similarly, in certain embodiments, the amino acid at position 54 ( . . .VILYDGSNKYY . . . in 11F11) can be substituted with S or E, or if theamino acid at this position is S, then the amino acid can be substitutedwith E. Antibodies 11F11, 73.4, 73.5, 24H2, 10D2, and 5F8 have a G atthis position, antibodies 73.6-73.9, 6E11, 7A11, 4C3, and 73.3 have a Sat this position, and antibodies 73.10 and 4D4 have an E at thisposition.

Other permissible substitutions in the variable region can be determinedbased on the alignment of the heavy and light chain variable regionsequences in FIG. 35 using a similar rationale as described above.

Antibodies having sequences with homology to those of CD73.3, CD73.4,CD73.5, CD73.6, CD73.7, CD73.8, CD73.9, CD73.10, CD73.11, 11F11, 4C3,4D4, 10D2, 11A6, 24H2, 5F8, 6E11 and/or 7A11, e.g., the VH and VLregions of SEQ ID NOs: 4, 16, 32, 40, 52, 60, 68, 80, 88, 135, 170-177,and SEQ ID NOs: 8, 12, 20, 24, 28, 36, 44, 48, 56, 64, 72, 76, 84, 92,respectively, or heavy and light chains of SEQ ID NOs: 100, 103, 107,109, 112, 114, 116, 119, 121, 133, and 184-210, and SEQ ID NOs: 101,102, 104, 105, 106, 108, 110, 111, 113, 115, 117, 118, 120 and 122,respectively, or CDRs can be obtained by mutagenesis (e.g.,site-directed or PCR-mediated mutagenesis) of nucleic acid molecules,e.g., SEQ ID NOs: 139, 142, 146, 148, 151, 153, 155, 158, 160, 237and/or SEQ ID NOs: 140, 141, 143, 144, 145, 147, 149, 150, 152, 154,156, 157, 159, 161 or SEQ ID NOs: 134, 243, 246, 250, 252, 255, 257,259, 262, 264, and/or SEQ ID NOs: 244, 245, 247, 248, 249, 251, 253,254, 256, 258, 260, 261, 263, 265, 266 followed by testing of theencoded altered antibody for retained function using the functionalassays described herein.

V. Antibodies with Conservative Modifications

Anti-CD73 antibodies may comprise a heavy chain variable regioncomprising CDR1, CDR2 and CDR3 sequences and a light chain variableregion comprising CDR1, CDR2 and CDR3 sequences, wherein one or more ofthese CDR sequences comprise specified amino acid sequences based on thepreferred antibodies described herein e.g., of CD73.4-1, CD73.4-2,CD73.3, 11F11-1, 11F11-2, 4C3-1, 4C3-2, 4C3-3, 4D4, 10D2-1, 10D2-2,11A6, 24H2, 5F8-1, 5F8-2, 6E11 and/or 7A11, or conservativemodifications thereof, and wherein the antibodies retain the desiredfunctional properties of the anti-CD73 antibodies described herein.Accordingly, an isolated anti-CD73 antibody, or antigen binding portionthereof, may comprise a heavy chain variable region comprising CDR1,CDR2, and CDR3 sequences and a light chain variable region comprisingCDR1, CDR2, and CDR3 sequences, wherein:

-   -   (a) the heavy chain variable region CDR3 sequence comprises an        amino acid sequence selected from the group consisting of amino        acid sequences of SEQ ID NOs: 7, 19, 35, 43, 55, 63, 71, 83, and        91, and conservative modifications thereof, e.g., 1, 2, 3, 4, 5,        1-2, 1-3, 1-4 or 1-5 conservative amino acid substitutions;    -   (b) the light chain variable region CDR3 sequence comprises an        amino acid sequence selected from the group consisting of amino        acid sequence of SEQ ID NOs: 11, 15, 23, 27, 31, 39, 47, 51, 59,        67, 75, 79, 87, and 95, and conservative modifications thereof,        e.g., 1, 2, 3, 4, 5, 1-2, 1-3, 1-4 or 1-5 conservative amino        acid substitutions;    -   (c) the antibody specifically binds to CD73, and    -   (d) the antibody exhibits 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all        of the functional properties listed in Table 3.

In a preferred embodiment, the heavy chain variable region CDR2 sequencecomprises an amino acid sequence selected from the group consisting ofamino acid sequences of SEQ ID NOs: 6, 18, 34, 42, 54, 62, 70, 82, and90, and conservative modifications thereof, e.g., 1, 2, 3, 4, 5, 1-2,1-3, 1-4 or 1-5 conservative amino acid substitutions; and the lightchain variable region CDR2 sequence comprises an amino acid sequenceselected from the group consisting of amino acid sequences of SEQ IDNOs: 10, 14, 22, 26, 30, 38, 46, 50, 58, 66, 74, 78, 86, and 94, andconservative modifications thereof, e.g., 1, 2, 3, 4, 5, 1-2, 1-3, 1-4or 1-5 conservative amino acid substitutions. In another preferredembodiment, the heavy chain variable region CDR1 sequence comprises anamino acid sequence selected from the group consisting of amino acidsequences of SEQ ID NOs: 5, 17, 33, 41, 53, 61, 69, 81, and 89, andconservative modifications thereof, e.g., 1, 2, 3, 4, 5, 1-2, 1-3, 1-4or 1-5 conservative amino acid substitutions; and the light chainvariable region CDR1 sequence comprises an amino acid sequence selectedfrom the group consisting of amino acid sequences of SEQ ID NOs: 9, 13,21, 25, 29, 37, 45, 49, 57, 65, 73, 77, 85, and 93, and conservativemodifications thereof, e.g., 1, 2, 3, 4, 5, 1-2, 1-3, 1-4 or 1-5conservative amino acid substitutions.

In various embodiments, the antibodies can be, for example, humanantibodies, humanized antibodies or chimeric antibodies.

Conservative amino acid substitutions may also be made in portions ofthe antibodies other than, or in addition to, the CDRs. For example,conservative amino acid modifications may be made in a framework regionor in the constant region, e.g., Fc region. Any of the substitutionsdescribed herein may be a conservative substitution. A variable regionor a heavy or light chain may comprise 1, 2, 3, 4, 5, 1-2, 1-3, 1-4,1-5, 1-10, 1-15, 1-20, 1-25, or 1-50 conservative amino acidsubstitutions relative to the anti-CD73 antibody sequences providedherein. In certain embodiments, an anti-CD73 antibody comprises acombination of conservative and non-conservative amino acidmodification.

VI. Antibodies that Bind the Same Epitope on CD73 as or Compete forBinding to CD73 with the Antibodies Described Herein

Also provided are antibodies that compete for binding to CD73 with theparticular anti-CD73 antibodies described herein (e.g., antibodiesCD73.4, CD73.3, 11F11, 4C3, 4D4, 10D2, 11A6, 24H2, 5F8, 6E11 and 7A11).Such competing antibodies can be identified based on their ability tocompetitively inhibit binding to CD73 of one or more of monoclonalantibodies 11F11-1, 11F11-2, 4C3-1, 4C3-2, 4C3-3, 4D4, 10D2-1, 10D2-2,11A6, 24H2, 5F8-1, 5F8-2, 6E11 7A11 and/or CD73.3 or CD73.4 (with anyconstant regions and light chains described herein for these antibodies)in standard CD73 binding assays. For example, standard ELISA assays orcompetitive ELISA assays can be used in which a recombinant human CD73protein is immobilized on the plate, various concentrations of unlabeledfirst antibody are added, the plate is washed, labeled second antibodyis added, washed, and the amount of bound label is measured. If theincreasing concentration of the unlabeled (first) antibody (alsoreferred to as the “blocking antibody”) inhibits the binding of thelabeled (second) antibody, the first antibody is said to inhibit thebinding of the second antibody to the target on the plate, or is said tocompete with the binding of the second antibody. Additionally oralternatively, BIACORE® SPR analysis can be used to assess the abilityof the antibodies to compete. The ability of a test antibody to inhibitthe binding of an anti-CD73 antibody described herein to CD73demonstrates that the test antibody can compete with the antibody forbinding to CD73.

Also provided herein are anti-CD73 antibodies that inhibit the bindingof anti-CD73 antibodies described herein to CD73 on cells, e.g., tumorcells, by at least 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%and/or whose binding to CD73 on cells, e.g., tumor cells, is inhibitedby at least 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, e.g., asmeasured by ELISA or FACS, such as by using the assay described in thepreceding paragraph.

Antibodies that compete for binding with the anti-CD73 antibodiesdescribed herein may be identified by using art-known methods. Forexample, mice may be immunized with human CD73 as described herein,hybridomas produced, and the resulting monoclonal antibodies screenedfor the ability to compete with an antibody described herein for bindingto CD73. Mice can also be immunized with a smaller fragment of CD73containing the epitope to which the antibody binds. The epitope orregion comprising the epitope can be identified by, e.g., screening forbinding to a series of overlapping peptides spanning CD73.Alternatively, the method of Jespers et al., Biotechnology 12:899, 1994may be used to guide the selection of antibodies having the same epitopeand therefore similar properties to the an anti-CD73 antibody describedherein. Using phage display, first the heavy chain of the anti-CD73antibody is paired with a repertoire of (preferably human) light chainsto select a CD73-binding antibody, and then the new light chain ispaired with a repertoire of (preferably human) heavy chains to select a(preferably human) CD73-binding antibody having the same epitope orepitope region as an anti-CD73 antibody described herein. Alternativelyvariants of an antibody described herein can be obtained by mutagenesisof cDNA encoding the heavy and light chains of the antibody.

Techniques for determining antibodies that bind to the “same epitope onCD73” with the antibodies described herein include, for example, epitopemapping methods, such as x-ray analyses of crystals of antigen: antibodycomplexes, which provides atomic resolution of the epitope. Othermethods monitor the binding of the antibody to antigen fragments ormutated variations of the antigen where loss of binding due to amodification of an amino acid residue within the antigen sequence isoften considered an indication of an epitope component. In addition,computational combinatorial methods for epitope mapping can also beused. Methods may also rely on the ability of an antibody of interest toaffinity isolate specific short peptides (either in native threedimensional form or in denatured form) from combinatorial phage displaypeptide libraries. The peptides are then regarded as leads for thedefinition of the epitope corresponding to the antibody used to screenthe peptide library. For epitope mapping, computational algorithms havealso been developed which have been shown to map conformationaldiscontinuous epitopes.

Alanine scanning mutagenesis, as described by Cunningham and Wells(1989) Science 244: 1081-1085, or some other form of point mutagenesisof amino acid residues in CD73 may also be used to determine thefunctional epitope for an anti-CD73 antibody. Mutagenesis studies,however, may also reveal amino acid residues that are crucial to theoverall three-dimensional structure of CD73 but that are not directlyinvolved in antibody-antigen contacts, and thus other methods may benecessary to confirm a functional epitope determined using this method.

The epitope or epitope region (an “epitope region” is a regioncomprising the epitope or overlapping with the epitope) bound by aspecific antibody may also be determined by assessing binding of theantibody to peptides comprising fragments of CD73, e.g., non-denaturedor denatured fragments. A series of overlapping peptides encompassingthe sequence of CD73 (e.g., human CD73) may be synthesized and screenedfor binding, e.g. in a direct ELISA, a competitive ELISA (where thepeptide is assessed for its ability to prevent binding of an antibody toCD73 bound to a well of a microtiter plate), or on a chip. Such peptidescreening methods may not be capable of detecting some discontinuousfunctional epitopes, i.e. functional epitopes that involve amino acidresidues that are not contiguous along the primary sequence of the CD73polypeptide chain.

An epitope may also be identified by MS-based protein footprinting, suchas Hydrogen/deuterium exchange mass spectrometry (HDX-MS) and FastPhotochemical Oxidation of Proteins (FPOP). HDX-MS may be conducted,e.g., as further described in the Examples and in Wei et al. (2014) DrugDiscovery Today 19:95, the methods of which are specificallyincorporated by reference herein. FPOP may be conducted as described,e.g., in Hambley and Gross (2005) J. American Soc. Mass Spectrometry16:2057, the methods of which are specifically incorporated by referenceherein.

The epitope bound by anti-CD73 antibodies may also be determined bystructural methods, such as X-ray crystal structure determination (e.g.,WO2005/044853), molecular modeling and nuclear magnetic resonance (NMR)spectroscopy, including NMR determination of the H-D exchange rates oflabile amide hydrogens in CD73 when free and when bound in a complexwith an antibody of interest (Zinn-Justin et al. (1992) Biochemistry 31,11335-11347; Zinn-Justin et al. (1993) Biochemistry 32, 6884-6891).

With regard to X-ray crystallography, crystallization may beaccomplished using any of the known methods in the art (e.g. Giege etal. (1994) Acta Crystallogr. D50:339-350; McPherson (1990) Eur. J.Biochem. 189:1-23), including microbatch (e.g. Chayen (1997) Structure5:1269-1274), hanging-drop vapor diffusion (e.g. McPherson (1976) J.Biol. Chem. 251:6300-6303), seeding and dialysis. It is desirable to usea protein preparation having a concentration of at least about 1 mg/mLand preferably about 10 mg/mL to about 20 mg/mL. Crystallization may bebest achieved in a precipitant solution containing polyethylene glycol1000-20,000 (PEG; average molecular weight ranging from about 1000 toabout 20,000 Da), preferably about 5000 to about 7000 Da, morepreferably about 6000 Da, with concentrations ranging from about 10% toabout 30% (w/v). It may also be desirable to include a proteinstabilizing agent, e.g. glycerol at a concentration ranging from about0.5% to about 20%. A suitable salt, such as sodium chloride, lithiumchloride or sodium citrate may also be desirable in the precipitantsolution, preferably in a concentration ranging from about 1 mM to about1000 mM. The precipitant is preferably buffered to a pH of from about3.0 to about 5.0, preferably about 4.0. Specific buffers useful in theprecipitant solution may vary and are well-known in the art (Scopes,Protein Purification: Principles and Practice, Third ed., (1994)Springer-Verlag, New York). Examples of useful buffers include, but arenot limited to, HEPES, Tris, MES and acetate. Crystals may be grow at awide range of temperatures, including 2° C., 4° C., 8° C. and 26° C.

Antibody:antigen crystals may be studied using well-known X-raydiffraction techniques and may be refined using computer software suchas X-PLOR (Yale University, 1992, distributed by Molecular Simulations,Inc.; see e.g. Blundell & Johnson (1985) Meth. Enzymol. 114 & 115, H. W.Wyckoff et al., eds., Academic Press; U.S. Patent ApplicationPublication No. 2004/0014194), and BUSTER (Bricogne (1993) Acta Cryst.D49:37-60; Bricogne (1997) Meth. Enzymol. 276A:361-423, Carter & Sweet,eds.; Roversi et al. (2000) Acta Cryst. D56:1313-1323), the disclosuresof which are hereby incorporated by reference in their entireties.

Anti-CD73 antibodies may bind to the same epitope as any of theanti-CD73 antibodies having amino acid sequences described herein, asdetermined by an epitope mapping technique, such as a techniquedescribed herein. Anti-CD73 antibodies may also have similarinteractions with human CD73, e.g., they may have at least about 50%,60%, 70%, 80%, 90%, 95% or more of the interactions shown in Table 30,as determined by X-ray crystallography.

VII. Engineered and Modified Antibodies VH and VL Regions

Also provided are engineered and modified antibodies that can beprepared using an antibody having one or more of the V_(H) and/or V_(L)sequences disclosed herein as starting material to engineer a modifiedantibody, which modified antibody may have altered properties from thestarting antibody. An antibody can be engineered by modifying one ormore residues within one or both variable regions (i.e., V_(H) and/orV_(L)), for example within one or more CDR regions and/or within one ormore framework regions. Additionally or alternatively, an antibody canbe engineered by modifying residues within the constant region(s), forexample to alter the effector function(s) of the antibody.

One type of variable region engineering that can be performed is CDRgrafting. Antibodies interact with target antigens predominantly throughamino acid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificreference antibodies by constructing expression vectors that include CDRsequences from the specific reference antibody grafted onto frameworksequences from a different antibody with different properties (see,e.g., Riechmann, L. et al. (1998) Nature 332:323-327; Jones, P. et al.(1986) Nature 321:522-525; Queen, C. et al. (1989) Proc. Natl. Acad.See. U.S.A. 86:10029-10033; U.S. Pat. No. 5,225,539 to Winter, and U.S.Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.)

Accordingly, another embodiment described herein pertains to an isolatedmonoclonal antibody, or antigen binding portion thereof, comprising aheavy chain variable region comprising CDR1, CDR2, and CDR3 sequencescomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 5, 17, 33, 41, 53, 61, 69, 81, and 89, SEQ ID NOs: 6, 18,34, 42, 54, 62, 70, 82, and 90, and SEQ ID NOs: 7, 19, 35, 43, 55, 63,71, 83, and 91, respectively, and a light chain variable regioncomprising CDR1, CDR2, and CDR3 sequences comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 9, 13, 21,25, 29, 37, 45, 49, 57, 65, 73, 77, 85, and 93, SEQ ID NOs: 10, 14, 22,26, 30, 38, 46, 50, 58, 66, 74, 78, 86, and 94, and SEQ ID NOs:11, 15,23, 27, 31, 39, 47, 51, 59, 67, 75, 79, 87, and 95, respectively. Thus,such antibodies contain the V_(H) and V_(L) CDR sequences of monoclonalantibodies CD73.4-1, CD73.4-2, 11F11-1, 11F11-2, 4C3-1, 4C3-2, 4C3-3,4D4, 10D2-1, 10D2-2, 11A6, 24H2, 5F8-1, 5F8-2, 6E11 and 7A11, yet maycontain different framework sequences from these antibodies.

Such framework sequences can be obtained from public DNA databases orpublished references that include germline antibody gene sequences. Forexample, germline DNA sequences for human heavy and light chain variableregion genes can be found in the “VBase” human germline sequencedatabase (available on the Internet at www.mrc-cpe.cam.ac.uk/vbase), aswell as in Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242; Tomlinson, I. M., et al.(1992) “The Repertoire of Human Germline V_(H) Sequences Reveals aboutFifty Groups of V_(H) Segments with Different Hypervariable Loops” J.Mol. Biol. 227:776-798; and Cox, J. P. L. et al. (1994) “A Directory ofHuman Germ-line V_(H) Segments Reveals a Strong Bias in their Usage”Eur. J. Immunol. 24:827-836; the contents of each of which are expresslyincorporated herein by reference.

Preferred framework sequences for use in the antibodies described hereinare those that are structurally similar to the framework sequences usedby antibodies described herein. The V_(H) CDR1, 2 and 3 sequences, andthe V_(L) CDR1, 2 and 3 sequences, can be grafted onto framework regionsthat have the identical sequence as that found in the germlineimmunoglobulin gene from which the framework sequence derive, or the CDRsequences can be grafted onto framework regions that contain up to 20,preferably conservative, amino acid substitutions as compared to thegermline sequences. For example, it has been found that in certaininstances it is beneficial to mutate residues within the frameworkregions to maintain or enhance the antigen binding ability of theantibody (see e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and6,180,370 to Queen et al).

Engineered antibodies described herein include those in whichmodifications have been made to framework residues within V_(H) and/orV_(L), e.g. to improve the properties of the antibody. Typically suchframework modifications are made to decrease the immunogenicity of theantibody. For example, one approach is to “backmutate” one or moreframework residues to the corresponding germline sequence. Morespecifically, an antibody that has undergone somatic mutation maycontain framework residues that differ from the germline sequence fromwhich the antibody is derived. Such residues can be identified bycomparing the antibody framework sequences to the germline sequencesfrom which the antibody is derived. To return the framework regionsequences to their germline configuration, the somatic mutations can be“backmutated” to the germline sequence by, for example, site-directedmutagenesis or PCR-mediated mutagenesis. Such “backmutated” antibodiesare also intended to be encompassed. Another type of frameworkmodification involves mutating one or more residues within the frameworkregion, or even within one or more CDR regions, to remove T cellepitopes to thereby reduce the potential immunogenicity of the antibody.This approach is also referred to as “deimmunization” and is describedin further detail in U.S. Patent Publication No. 20030153043 by Carr etal.

Another type of variable region modification is to mutate amino acidresidues within the CDR regions to improve one or more bindingproperties (e.g., affinity) of the antibody of interest. Site-directedmutagenesis or PCR-mediated mutagenesis can be performed to introducethe mutation(s) and the effect on antibody binding, or other functionalproperty of interest, can be evaluated in in vitro or in vivo assays asdescribed herein and provided in the Examples. Preferably conservativemodifications (as discussed above) are introduced. The mutations may beamino acid additions, deletions, or preferably substitutions. Moreover,typically no more than one, two, three, four or five residues within aCDR region are altered.

Accordingly, also provided are isolated anti-CD73 monoclonal antibodies,or antigen binding portions thereof, comprising a heavy chain variableregion comprising: (a) a V_(H) CDR1 region comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 5, 17, 33,41, 53, 61, 69, 81, and 89, or an amino acid sequence having one, two,three, four or five amino acid substitutions, deletions or additions ascompared to SEQ ID NOs: 5, 17, 33, 41, 53, 61, 69, 81, and 89; (b) aV_(H) CDR2 region comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 6, 18, 34, 42, 54, 62, 70, 82, and 90,or an amino acid sequence having one, two, three, four or five aminoacid substitutions, deletions or additions as compared to SEQ ID NOs: 6,18, 34, 42, 54, 62, 70, 82, and 90; (c) a V_(H) CDR3 region comprisingan amino acid sequence selected from the group consisting of SEQ IDNOs:7, 19, 35, 43, 55, 63, 71, 83, and 91, or an amino acid sequencehaving one, two, three, four or five amino acid substitutions, deletionsor additions as compared to SEQ ID NOs: 7, 19, 35, 43, 55, 63, 71, 83,and 91; (d) a V_(L) CDR1 region comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 9, 13, 21, 25, 29, 37,45, 49, 57, 65, 73, 77, 85, and 93, or an amino acid sequence havingone, two, three, four or five amino acid substitutions, deletions oradditions as compared to SEQ ID NOs: 9, 13, 21, 25, 29, 37, 45, 49, 57,65, 73, 77, 85, and 93; (e) a V_(L) CDR2 region comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 10, 14, 22,26, 30, 38, 46, 50, 58, 66, 74, 78, 86, and 94, or an amino acidsequence having one, two, three, four or five amino acid substitutions,deletions or additions as compared to SEQ ID NOs: 10, 14, 22, 26, 30,38, 46, 50, 58, 66, 74, 78, 86, and 94; and (f) a V_(L) CDR3 regioncomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 11, 15, 23, 27, 31, 39, 47, 51, 59, 67, 75, 79, 87, and 95,or an amino acid sequence having one, two, three, four or five aminoacid substitutions, deletions or additions as compared to SEQ ID NOs:11, 15, 23, 27, 31, 39, 47, 51, 59, 67, 75, 79, 87, and 95.

Methionine residues in CDRs of antibodies can be oxidized, resulting inpotential chemical degradation and consequent reduction in potency ofthe antibody. Accordingly, also provided are anti-CD73 antibodies thathave one or more methionine residues in the heavy and/or light chainCDRs replaced with amino acid residues that do not undergo oxidativedegradation.

Similarly, deamidation sites may be removed from anti-CD73 antibodies,particularly in the CDRs.

Potential glycosylation sites within the antigen binding domain arepreferably eliminated to prevent glycosylation that may interfere withantigen binding. See, e.g., U.S. Pat. No. 5,714,350.

Targeted Antigen Binding

In various embodiments, the antibody of the present invention ismodified to selectively block antigen binding in tissues andenvironments where antigen binding would be detrimental, but allowantigen binding where it would be beneficial. In one embodiment, ablocking peptide “mask” is generated that specifically binds to theantigen binding surface of the antibody and interferes with antigenbinding, which mask is linked to each of the binding arms of theantibody by a peptidase cleavable linker. See, e.g., U.S. Pat. No.8,518,404 to CytomX. Such constructs are useful for treatment of cancersin which protease levels are greatly increased in the tumormicroenvironment compared with non-tumor tissues. Selective cleavage ofthe cleavable linker in the tumor microenvironment allows disassociationof the masking/blocking peptide, enabling antigen binding selectively inthe tumor, rather than in peripheral tissues in which antigen bindingmight cause unwanted side effects.

Alternatively, in a related embodiment, a bivalent binding compound(“masking ligand”) comprising two antigen binding domains is developedthat binds to both antigen binding surfaces of the (bivalent) antibodyand interfere with antigen binding, in which the two binding domainsmasks are linked to each other (but not the antibody) by a cleavablelinker, for example cleavable by a peptidase. See, e.g., Intl Pat. App.Pub. No. WO 2010/077643 to Tegopharm Corp. Masking ligands may comprise,or be derived from, the antigen to which the antibody is intended tobind, or may be independently generated. Such masking ligands are usefulfor treatment of cancers in which protease levels are greatly increasedin the tumor microenvironment compared with non-tumor tissues. Selectivecleavage of the cleavable linker in the tumor microenvironment allowsdisassociation of the two binding domains from each other, reducing theavidity for the antigen-binding surfaces of the antibody. The resultingdissociation of the masking ligand from the antibody enables antigenbinding selectively in the tumor, rather than in peripheral tissues inwhich antigen binding might cause unwanted side effects.

Fcs and Modified Fcs

In addition to the activity of a therapeutic antibody arising frombinding of the antigen binding domain to the antigen (e.g. blocking of acognate ligand or receptor protein in the case of antagonist antibodies,or induced signaling in the case of agonist antibodies), the Fc portionof the antibody interact with the immune system generally in complexways to elicit any number of biological effects. Effector functions,such as The Fc region of an immunoglobulin is responsible for manyimportant antibody functions, such as antigen-dependent cellularcytotoxicity (ADCC), complement dependent cytotoxicity (CDC), andantibody-dependent cell-mediated phagocytosis (ADCP), result in killingof target cells, albeit by different mechanisms.

Anti-CD73 antibodies may comprise the variable domains of the antibodiesdescribed herein with constant domains comprising different Fc regions,selected based on the biological activities (if any) of the antibody forthe intended use. Salfeld (2007) Nat. Biotechnol. 25:1369. Human IgGs,for example, can be classified into four subclasses, IgG1, IgG2, IgG3,and IgG4, and each these of these comprises an Fc region having a uniqueprofile for binding to one or more of Fcγ receptors (activatingreceptors FcγRI (CD64), FcγRIIA, FcγRIIC (CD32); FcγRIIIA and FcγRIIIB(CD16) and inhibiting receptor FcγRIIB), and for the first component ofcomplement (C1q). Human IgG1 and IgG3 bind to all Fcγ receptors; IgG2binds to FcγRIIA_(H131), and with lower affinity to FcγRIIA_(R131)FcγRIIIA_(V158); IgG4 binds to FcγRI, FcγRIIA, FcγRIIB, FcγRIIC, andFcγRIIIA_(V158); and the inhibitory receptor FcγRIIB has a loweraffinity for IgG1, IgG2 and IgG3 than all other Fcγ receptors. Bruhns etal. (2009) Blood 113:3716. Studies have shown that FcγRI does not bindto IgG2, and FcγRIIIB does not bind to IgG2 or IgG4. Id. In general,with regard to ADCC activity, human IgG1≥IgG3>>IgG4≥IgG2. As aconsequence, for example, an IgG1 constant domain, rather than an IgG2or IgG4, might be chosen for use in a drug where ADCC is desired; IgG3might be chosen if activation of FcγRIIIA-expressing NK cells, monocytesof macrophages; and IgG4 might be chosen if the antibody is to be usedto desensitize allergy patients. IgG4 may also be selected if it isdesired that the antibody lack all effector function.

Accordingly, anti-CD73 variable regions described herein may be linked(e.g., covalently linked or fused) to an Fc, e.g., an IgG1, IgG2, IgG3or IgG4 Fc, which may be of any allotype or isoallotype, e.g., for IgG1:G1m, G1m1(a), G1m2(x), G1m3(f), G1m17(z); for IgG2: G2m, G2m23(n); forIgG3: G3m, G3m21(g1), G3m28(g5), G3m11(b0), G3m5(b1), G3m13(b3),G3m14(b4), G3m10(b5), G3m15(s), G3m16(t), G3m6(c3), G3m24(c5), G3m26(u),G3m27(v). See, e.g., Jefferis et al. (2009) mAbs 1:1). Selection ofallotype may be influenced by the potential immunogenicity concerns,e.g. to minimize the formation of anti-drug antibodies.

Variable regions described herein may be linked to an Fc comprising oneor more modifications, typically to alter one or more functionalproperties of the antibody, such as serum half-life, complementfixation, Fc receptor binding, and/or antigen-dependent cellularcytotoxicity. Furthermore, an antibody described herein may bechemically modified (e.g., one or more chemical moieties can be attachedto the antibody) or it may be modified to alter its glycosylation, toalter one or more functional properties of the antibody. Each of theseembodiments is described in further detail below. The numbering ofresidues in the Fc region is that of the EU index of Kabat. Sequencevariants disclosed herein are provided with reference to the residuenumber followed by the amino acid that is substituted in place of thenaturally occurring amino acid, optionally preceded by the naturallyoccurring residue at that position. Where multiple amino acids may bepresent at a given position, e.g. if sequences differ between naturallyoccurring isotypes, or if multiple mutations may be substituted at theposition, they are separated by slashes (e.g. “X/Y/Z”).

For example, one may make modifications in the Fc region in order togenerate an Fc variant with (a) increased or decreasedantibody-dependent cell-mediated cytotoxicity (ADCC), (b) increased ordecreased complement mediated cytotoxicity (CDC), (c) increased ordecreased affinity for C1q and/or (d) increased or decreased affinityfor a Fc receptor relative to the parent Fc. Such Fc region variantswill generally comprise at least one amino acid modification in the Fcregion. Combining amino acid modifications is thought to be particularlydesirable. For example, the variant Fc region may include two, three,four, five, etc substitutions therein, e.g. of the specific Fc regionpositions identified herein. Exemplary Fc sequence variants aredisclosed herein, and are also provided at U.S. Pat. Nos. 5,624,821;6,277,375; 6,737,056; 6,194,551; 7,317,091; 8,101.720; PCT PatentPublications WO 00/42072; WO 01/58957; WO 04/016750; WO 04/029207; WO04/035752; WO 04/074455; WO 04/099249; WO 04/063351; WO 05/070963: WO05/040217, WO 05/092925 and WO 06/020114.

Reducing Effector Function

ADCC activity may be reduced by modifying the Fc region. In certainembodiments, sites that affect binding to Fc receptors may be removed,preferably sites other than salvage receptor binding sites. In otherembodiments, an Fc region may be modified to remove an ADCC site. ADCCsites are known in the art; see, for example, Sarmay et al. (1992)Molec. Immunol. 29 (5): 633-9 with regard to ADCC sites in IgG1. In oneembodiment, the G236R and L328R variant of human IgG1 effectivelyeliminates FcγR binding. Horton et al. (2011) J. Immunol. 186:4223 andChu et al. (2008) Mol. Immunol. 45:3926. In other embodiments, the Fchaving reduced binding to FcγRs comprised the amino acid substitutionsL234A, L235E and G237A. Gross et al. (2001) Immunity 15:289.

CDC activity may also be reduced by modifying the Fc region. Mutationsat IgG1 positions D270, K322, P329 and P331, specifically alaninemutations D270A, K322A, P329A and P331A, significantly reduce theability of the corresponding antibody to bind C1q and activatecomplement. Idusogie et al. (2000) J. Immunol. 164:4178; WO 99/51642.Modification of position 331 of IgG1 (e.g. P331S) has been shown toreduce complement binding. Tao et al. (1993) J. Exp. Med. 178:661 andCanfield & Morrison (1991) J. Exp. Med. 173:1483. In another example,one or more amino acid residues within amino acid positions 231 to 239are altered to thereby reduce the ability of the antibody to fixcomplement. WO 94/29351.

In some embodiments, the Fc with reduced complement fixation has theamino acid substitutions A330S and P331S. Gross et al. (2001) Immunity15:289.

For uses where effector function is to be avoided altogether, e.g. whenantigen binding alone is sufficient to generate the desired therapeuticbenefit, and effector function only leads to (or increases the risk of)undesired side effects, IgG4 antibodies may be used, or antibodies orfragments lacking the Fc region or a substantial portion thereof can bedevised, or the Fc may be mutated to eliminate glycosylation altogether(e.g. N297A). Alternatively, a hybrid construct of human IgG2 (CH1domain and hinge region) and human IgG4 (CH2 and CH3 domains) has beengenerated that is devoid of effector function, lacking the ability tobind the FcγRs (like IgG2) and unable to activate complement (likeIgG4). Rother et al. (2007) Nat. Biotechnol. 25:1256. See also Muelleret al. (1997) Mol. Immunol. 34:441; Labrijn et al. (2008) Curr. Op.Immunol. 20:479 (discussing Fc modifications to reduce effector functiongenerally).

In other embodiments, the Fc region is altered by replacing at least oneamino acid residue with a different amino acid residue to reduce alleffector function(s) of the antibody. For example, one or more aminoacids selected from amino acid residues 234, 235, 236, 237, 297, 318,320 and 322 can be replaced with a different amino acid residue suchthat the antibody has decreased affinity for an effector ligand butretains the antigen-binding ability of the parent antibody. The effectorligand to which affinity is altered can be, for example, an Fc receptor(residues 234, 235, 236, 237, 297) or the C1 component of complement(residues 297, 318, 320, 322). U.S. Pat. Nos. 5,624,821 and 5,648,260,both by Winter et al.

WO 88/007089 proposed modifications in the IgG Fc region to decreasebinding to FcγRI to decrease ADCC (234A; 235E; 236A; G237A) or blockbinding to complement component C1q to eliminate CDC (E318A or V/K320Aand K322A/Q). See also Duncan & Winter (1988) Nature 332:563; Chappel etal. (1991) Proc. Nat'l Acad. Sci. (USA) 88:9036; and Sondermann et al.(2000) Nature 406:267 (discussing the effects of these mutations onFcγRIII binding).

Fc modifications reducing effector function also include substitutions,insertions, and deletions at positions 234, 235, 236, 237, 267, 269,325, and 328, such as 234G, 235G, 236R, 237K, 267R, 269R, 325L, and328R. An Fc variant may comprise 236R/328R. Other modifications forreducing FcγR and complement interactions include substitutions 297A,234A, 235A, 237A, 318A, 228P, 236E, 268Q, 309L, 330S, 331S, 220S, 226S,229S, 238S, 233P, and 234V. These and other modifications are reviewedin Strohl (2009) Current Opinion in Biotechnology 20:685-691. Effectorfunctions (both ADCC and complement activation) can be reduced, whilemaintaining neonatal FcR binding (maintaining half-life), by mutatingIgG residues at one or more of positions 233-236 and 327-331, such asE233P, L234V, L235A, optionally G236Δ, A327G, A330S and P331S in IgG1;E233P, F234V, L235A, optionally G2364 in IgG4; and A330S and P331S inIgG2. See Armour et al. (1999) Eur. J. Immunol. 29:2613; WO 99/58572.Other mutations that reduce effector function include L234A and L235A inIgG1 (Alegre et al. (1994) Transplantation 57:1537); V234A and G237A inIgG2 (Cole et al. (1997) J. Immunol. 159:3613; see also U.S. Pat. No.5,834,597); and S228P and L235E for IgG4 (Reddy et al. (2000) J.Immunol. 164:1925). Another combination of mutations for reducingeffector function in a human IgG1 include L234F, L235E and P331S.Oganesyan et al. (2008) Acta Crystallogr. D. Biol. Crystallogr. 64:700.See generally Labrijn et gal. (2008) Curr. Op. Immunol. 20:479.Additional mutations found to decrease effector function in the contextof an Fc (IgG1) fusion protein (abatacept) are C226S, C229S and P238S(EU residue numbering). Davis et al. (2007) J. Immunol. 34:2204.

Other Fc variants having reduced ADCC and/or CDC are disclosed atGlaesner et al. (2010) Diabetes Metab. Res. Rev. 26:287 (F234A and L235Ato decrease ADCC and ADCP in an IgG4); Hutchins et al. (1995) Proc.Nat'l Acad. Sci. (USA) 92:11980 (F234A, G237A and E318A in an IgG4); Anet al. (2009) MAbs 1:572 and U.S. Pat. App. Pub. 2007/0148167 (H268Q,V309L, A330S and P331S in an IgG2); McEarchern et al. (2007) Blood109:1185 (C226S, C229S, E233P, L234V, L235A in an IgG1); Vafa et al.(2014) Methods 65:114 (V234V, G237A, P238S, H268A, V309L, A330S, P331Sin an IgG2).

In certain embodiments, an Fc is chosen that has essentially no effectorfunction, i.e., it has reduced binding to FcγRs and reduced complementfixation. An exemplary Fc, e.g., IgG1 Fc, that is effectorless comprisesthe following five mutations: L234A, L235E, G237A, A330S and P331S.Gross et al. (2001) Immunity 15:289. Exemplary heavy chains comprisingthese mutations are set forth in the Sequence Listing, as detailed atTable 35. These five substitutions may be combined with N297A toeliminate glycosylation as well.

Enhancing Effector Function

Alternatively, ADCC activity may be increased by modifying the Fcregion. With regard to ADCC activity, human IgG1≥IgG3>>IgG4≥IgG2, so anIgG1 constant domain, rather than an IgG2 or IgG4, might be chosen foruse in a drug where ADCC is desired. Alternatively, the Fc region may bemodified to increase antibody dependent cellular cytotoxicity (ADCC)and/or to increase the affinity for an Fcγ receptor by modifying one ormore amino acids at the following positions: 234, 235, 236, 238, 239,240, 241, 243, 244, 245, 247, 248, 249, 252, 254, 255, 256, 258, 262,263, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286,289, 290, 292, 293, 294, 295, 296, 298, 299, 301, 303, 305, 307, 309,312, 313, 315, 320, 322, 324, 325, 326, 327, 329, 330, 331, 332, 333,334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414,416, 419, 430, 433, 434, 435, 436, 437, 438 or 439. See WO 2012/142515;see also WO 00/42072. Exemplary substitutions include 236A, 239D, 239E,268D, 267E, 268E, 268F, 324T, 332D, and 332E. Exemplary variants include239D/332E, 236A/332E, 236A/239D/332E, 268F/324T, 267E/268F, 267E/324T,and 267E/268F/324T. For example, human IgG1Fcs comprising the G236Avariant, which can optionally be combined with 1332E, have been shown toincrease the FcγIIA/FcγIIB binding affinity ratio approximately 15-fold.Richards et al. (2008) Mol. Cancer Therap. 7:2517; Moore et al. (2010)mAbs 2:181. Other modifications for enhancing FcyR and complementinteractions include but are not limited to substitutions 298A, 333A,334A, 326A, 247I, 339D, 339Q, 280Q, 290S, 298D, 298V, 243L, 292P, 300L,396L, 305I, and 396L. These and other modifications are reviewed inStrohl (2009) Current Opinion in Biotechnology 20:685-691. Specifically,both ADCC and CDC may be enhanced by changes at position E333 of IgG1,e.g. E333A. Shields et al. (2001) J. Biol. Chem. 276:6591. The use ofP247I and A339D/Q mutations to enhance effector function in an IgG1 isdisclosed at WO 2006/020114, and D280H, K290S±S298D/V is disclosed at WO2004/074455. The K326A/W and E333A/S variants have been shown toincrease effector function in human IgG1, and E333S in IgG2. Idusogie etal. (2001) J. Immunol. 166:2571.

Specifically, the binding sites on human IgG1 for FcγR1, FcγRII, FcγRIIIand FcRn have been mapped, and variants with improved binding have beendescribed. Shields et al. (2001) J. Biol. Chem. 276:6591-6604. Specificmutations at positions 256, 290, 298, 333, 334 and 339 were shown toimprove binding to FcγRIII, including the combination mutantsT256A/S298A, S298A/E333A, S298A/K224A and S298A/E333A/K334A (havingenhanced FcγRIIIa binding and ADCC activity). Other IgG1 variants withstrongly enhanced binding to FcγRIIIa have been identified, includingvariants with S239D/I332E and S239D/I332E/A330L mutations which showedthe greatest increase in affinity for FcγRIIIa, a decrease in FcγRIIbbinding, and strong cytotoxic activity in cynomolgus monkeys. Lazar etal. (2006) Proc. Nat'l Acad Sci. (USA) 103:4005; Awan et al. (2010)Blood 115:1204; Desjarlais & Lazar (2011) Exp. Cell Res. 317:1278.Introduction of the triple mutations into antibodies such as alemtuzumab(CD52-specific), trastuzumab (HER2/neu-specific), rituximab(CD20-specific), and cetuximab (EGFR-specific) translated into greatlyenhanced ADCC activity in vitro, and the S239D/I332E variant showed anenhanced capacity to deplete B cells in monkeys. Lazar et al. (2006)Proc. Nat'l Acad Sci. (USA) 103:4005. In addition, IgG1 mutantscontaining L235V, F243L, R292P, Y300L, V3051 and P396L mutations whichexhibited enhanced binding to FcγRIIIa and concomitantly enhanced ADCCactivity in transgenic mice expressing human FcγRIIIa in models of Bcell malignancies and breast cancer have been identified. Stavenhagen etal. (2007) Cancer Res. 67:8882; U.S. Pat. No. 8,652,466; Nordstrom etal. (2011) Breast Cancer Res. 13:R123.

Different IgG isotypes also exhibit differential CDC activity(IgG3>IgG1>>IgG2≈IgG4). Dangl et al. (1988) EMBO J. 7:1989. For uses inwhich enhanced CDC is desired, it is also possible to introducemutations that increase binding to C1q. The ability to recruitcomplement (CDC) may be enhanced by mutations at K326 and/or E333 in anIgG2, such as K326W (which reduces ADCC activity) and E333S, to increasebinding to C1q, the first component of the complement cascade. Idusogieet al. (2001) J. Immunol. 166:2571. Introduction of S267E/H268F/S324T(alone or in any combination) into human IgG1 enhances C1q binding.Moore et al. (2010) mAbs 2:181. The Fc region of the IgG1/IgG3 hybridisotype antibody “113F” of Natsume et al. (2008) Cancer Res. 68:3863(FIG. 1 therein) also confers enhanced CDC. See also Michaelsen et al.(2009) Scand. J. Immunol. 70:553 and Redpath et al. (1998) Immunology93:595.

Additional mutations that can increase or decrease effector function aredisclosed at Dall'Acqua et al. (2006) J. Immunol. 177:1129. See alsoCarter (2006) Nat. Rev. Immunol. 6:343; Presta (2008) Curr. Op. Immunol.20:460.

Fc variants that enhance affinity for the inhibitory receptor FcγRIIbmay also be used, e.g. to enhance apoptosis-inducing or adjuvantactivity. Li & Ravetch (2011) Science 333:1030; Li & Ravetch (2012)Proc. Nat'l Acad. Sci (USA) 109:10966; U.S. Pat. App. Pub. 2014/0010812.Such variants may provide an antibody with immunomodulatory activitiesrelated to FcyRllb⁺ cells, including for example B cells and monocytes.In one embodiment, the Fc variants provide selectively enhanced affinityto FcyRllb relative to one or more activating receptors. Modificationsfor altering binding to FcyRllb include one or more modifications at aposition selected from the group consisting of 234, 235, 236, 237, 239,266, 267, 268, 325, 326, 327, 328, and 332, according to the EU index.Exemplary substitutions for enhancing FcyRllb affinity include but arenot limited to 234D, 234E, 234F, 234W, 235D, 235F, 235R, 235Y, 236D,236N, 237D, 237N, 239D, 239E, 266M, 267D, 267E, 268D, 268E, 327D, 327E,328F, 328W, 328Y, and 332E. Exemplary substitutions include 235Y, 236D,239D, 266M, 267E, 268D, 268E, 328F, 328W, and 328Y. Other Fc variantsfor enhancing binding to FcyRllb include 235Y/267E, 236D/267E,239D/268D, 239D/267E, 267E/268D, 267E/268E, and 267E/328F. Specifically,the S267E, G236D, S239D, L328F and I332E variants, including theS267E+L328F double variant, of human IgG1 are of particular value inspecifically enhancing affinity for the inhibitory FcyRllb receptor. Chuet al. (2008) Mol. Immunol. 45:3926; U.S. Pat. App. Pub. 2006/024298; WO2012/087928. Enhanced specificity for FcγRIIb (as distinguished fromFcγRIIa^(R131)) may be obtained by adding the P238D substitution. Mimotoet al. (2013) Protein. Eng. Des. & Selection 26:589; WO 2012/115241.

In certain embodiments, the antibody is modified to increase itsbiological half-life. Various approaches are possible. For example, thismay be done by increasing the binding affinity of the Fc region forFcRn. In one embodiment, the antibody is altered within the CH1 or CLregion to contain a salvage receptor binding epitope taken from twoloops of a CH2 domain of an Fc region of an IgG, as described in U.S.Pat. Nos. 5,869,046 and 6,121,022 by Presta et al. Other exemplary Fcvariants that increase binding to FcRn and/or improve pharmacokineticproperties include substitutions at positions 259, 308, and 434,including for example 2591, 308F, 428L, 428M, 434S, 434H, 434F, 434Y,and 434M. Other variants that increase Fc binding to FcRn include; 250E,250Q, 428L, 428F, 250Q/428L (Hinton et al., 2004, J. Biol. Chem. 279(8):6213-6216, Hinton et al. 2006 Journal of Immunology 176:346-356), 256A,272A, 305A, 307A, 31 1A, 312A, 378Q, 380A, 382A, 434A (Shields et al,Journal of Biological Chemistry, 2001, 276(9):6591-6604), 252F, 252Y,252W, 254T, 256Q, 256E, 256D, 433R, 434F, 434Y, 252Y/254T/256E,433K/434F/436H (Dall Acqua et al. Journal of Immunology, 2002,169:5171-5180, Dall'Acqua et al., 2006, Journal of Biological Chemistry281:23514-23524). See U.S. Pat. No. 8,367,805.

Modification of certain conserved residues in IgG Fc(1253/H310/Q311/H433/N434), such as the N434A variant (Yeung et al.(2009) J. Immunol. 182:7663), has been proposed as a way to increaseFcRn affinity, thus increasing the half-life of the antibody incirculation. WO 98/023289. The combination Fc variant comprising M428Land N434S has been shown to increase FcRn binding and increase serumhalf-life up to five-fold. Zalevsky et al. (2010) Nat. Biotechnol.28:157. The combination Fc variant comprising T307A, E380A and N434Amodifications also extends half-life of IgG1 antibodies. Petkova et al.(2006) Int. Immunol. 18:1759. In addition, combination Fc variantscomprising M252Y/M428L, M428L/N434H, M428L/N434F, M428L/N434Y,M428L/N434A, M428L/N434M, and M428L/N434S variants have also been shownto extend half-life. WO 2009/086320.

Further, a combination Fc variant comprising M252Y, S254T and T256E,increases half-life-nearly 4-fold. Dall'Acqua et al. (2006) J. Biol.Chem. 281:23514. A related IgG1 modification providing increased FcRnaffinity but reduced pH dependence (M252Y/S254T/T256E/H433K/N434F) hasbeen used to create an IgG1 construct (“MST-HN Abdeg”) for use as acompetitor to prevent binding of other antibodies to FcRn, resulting inincreased clearance of that other antibody, either endogenous IgG (e.g.in an autoimmune setting) or another exogenous (therapeutic) mAb.Vaccaro et al. (2005) Nat. Biotechnol. 23:1283; WO 2006/130834.

Other modifications for increasing FcRn binding are described in Yeunget al. (2010) J. Immunol. 182:7663-7671; 6,277,375; 6,821,505; WO97/34631; WO 2002/060919.

In certain embodiments, hybrid IgG isotypes may be used to increase FcRnbinding, and potentially increase half-life. For example, an IgG1/IgG3hybrid variant may be constructed by substituting IgG1 positions in theCH2 and/or CH3 region with the amino acids from IgG3 at positions wherethe two isotypes differ. Thus a hybrid variant IgG antibody may beconstructed that comprises one or more substitutions, e.g., 274Q, 276K,300F, 339T, 356E, 358M, 384S, 392N, 397M, 4221, 435R, and 436F. In otherembodiments described herein, an IgG1/IgG2 hybrid variant may beconstructed by substituting IgG2 positions in the CH2 and/or CH3 regionwith amino acids from IgG1 at positions where the two isotypes differ.Thus a hybrid variant IgG antibody may be constructed that comprises oneor more substitutions, e.g., one or more of the following amino acidsubstitutions: 233E, 234L, 235L, -236G (referring to an insertion of aglycine at position 236), and 327A. See U.S. Pat. No. 8,629,113. Ahybrid of IgG1/IgG2/IgG4 sequences has been generated that purportedlyincreases serum half-life and improves expression. U.S. Pat. No.7,867,491 (sequence number 18 therein).

The serum half-life of the antibodies of the present invention can alsobe increased by pegylation. An antibody can be pegylated to, forexample, increase the biological (e.g., serum) half-life of theantibody. To pegylate an antibody, the antibody, or fragment thereof,typically is reacted with a polyethylene glycol (PEG) reagent, such as areactive ester or aldehyde derivative of PEG, under conditions in whichone or more PEG groups become attached to the antibody or antibodyfragment. Preferably, the pegylation is carried out via an acylationreaction or an alkylation reaction with a reactive PEG molecule (or ananalogous reactive water-soluble polymer). As used herein, the term“polyethylene glycol” is intended to encompass any of the forms of PEGthat have been used to derivatize other proteins, such as mono (C1-C10)alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide.In certain embodiments, the antibody to be pegylated is an aglycosylatedantibody. Methods for pegylating proteins are known in the art and canbe applied to the antibodies described herein. See for example, EP0154316 by Nishimura et al. and EP 0401384 by Ishikawa et al.

Alternatively, under some circumstances it may be desirable to decreasethe half-life of an antibody of the present invention, rather thanincrease it. Modifications such as I253A (Hornick et al. (2000) J. Nucl.Med. 41:355) and H435A/R I253A or H310A (Kim et al. (2000) Eur. J.Immunol. 29:2819) in Fc of human IgG1 can decrease FcRn binding, thusdecreasing half-life (increasing clearance) for use in situations whererapid clearance is preferred, such a medical imaging. See also Kenanovaet al. (2005) Cancer Res. 65:622. Other means to enhance clearanceinclude formatting the antigen binding domains of the present inventionas antibody fragments lacking the ability to bind FcRn, such as Fabfragments. Such modification can reduce the circulating half-life of anantibody from a couple of weeks to a matter of hours.

Selective PEGylation of antibody fragments can then be used to fine-tune(increase) the half-life of the antibody fragments if necessary. Chapmanet al. (1999) Nat. Biotechnol. 17:780.

Antibody fragments may also be fused to human serum albumin, e.g. in afusion protein construct, to increase half-life. Yeh et al. (1992) Proc.Nat'l Acad. Sci. 89:1904. Alternatively, a bispecific antibody may beconstructed with a first antigen binding domain of the present inventionand a second antigen binding domain that binds to human serum albumin(HSA). See Int'l Pat. Appl. Pub. WO 2009/127691 and patent referencescited therein. Alternatively, specialized polypeptide sequences can beadded to antibody fragments to increase half-life, e.g. “XTEN”polypeptide sequences. Schellenberger et al. (2009) Nat. Biotechnol.27:1186; Int'l Pat. Appl. Pub. WO 2010/091122. Additional Fc Variants

When using an IgG4 constant domain, it is usually preferable to includethe substitution S228P, which mimics the hinge sequence in IgG1 andthereby stabilizes IgG4 molecules, e.g. reducing Fab-arm exchangebetween the therapeutic antibody and endogenous IgG4 in the patientbeing treated. Labrijn et al. (2009) Nat. Biotechnol. 27:767; Reddy etal. (2000) J Immunol. 164:1925.

A potential protease cleavage site in the hinge of IgG1 constructs canbe eliminated by D221G and K222S modifications, increasing the stabilityof the antibody. WO 2014/043344.

The affinities and binding properties of an Fc variant for its ligands(Fc receptors) may be determined by a variety of in vitro assay methods(biochemical or immunological based assays) known in the art includingbut not limited to, equilibrium methods (e.g., enzyme-linkedimmunoabsorbent assay (ELISA), or radioimmunoassay (RIA)), or kinetics(e.g., BIACORE® SPR analysis), and other methods such as indirectbinding assays, competitive inhibition assays, fluorescence resonanceenergy transfer (FRET), gel electrophoresis and chromatography (e.g.,gel filtration). These and other methods may utilize a label on one ormore of the components being examined and/or employ a variety ofdetection methods including but not limited to chromogenic, fluorescent,luminescent, or isotopic labels. A detailed description of bindingaffinities and kinetics can be found in Paul, W. E., ed., FundamentalImmunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), whichfocuses on antibody-immunogen interactions.

In still other embodiments, the glycosylation of an antibody is modifiedto increase or decrease effector function. For example, an aglycoslatedantibody can be made that lacks all effector function by mutating theconserved asparagine residue at position 297 (e.g. N297A), thusabolishing complement and FcγRI binding. Bolt et al. (1993) Eur. J.Immunol. 23:403. See also Tao & Morrison (1989) J. Immunol. 143:2595(using N297Q in IgG1 to eliminate glycosylation at position 297).

Although aglycosylated antibodies generally lack effector function,mutations can be introduced to restore that function. Aglycosylatedantibodies, e.g. those resulting from N297A/C/D/or H mutations orproduced in systems (e.g. E. coli) that do not glycosylate proteins, canbe further mutated to restore FcqR binding, e.g. S298G and/or T299A/G/orH (WO 2009/079242), or E382V and M428I (Jung et al. (2010) Proc. Nat'lAcad. Sci (USA) 107:604).

Additionally, an antibody with enhanced ADCC can be made by altering theglycosylation. For example, removal of fucose from heavy chainAsn297-linked oligosaccharides has been shown to enhance ADCC, based onimproved binding to FcγRIIIa. Shields et al. (2002) JBC 277:26733; Niwaet al. (2005) J. Immunol. Methods 306: 151; Cardarelli et al. (2009)Clin. Cancer Res. 15:3376 (MDX-1401); Cardarelli et al. (2010) CancerImmunol. Immunotherap. 59:257 (MDX-1342). Such low fucose antibodies maybe produced, e.g., in knockout Chinese hamster ovary (CHO) cells lackingfucosyltransferase (FUT8) (Yamane-Ohnuki et al. (2004) Biotechnol.Bioeng. 87:614), or in other cells that generate afucosylatedantibodies. See, e.g., Zhang et al. (2011) mAbs 3:289 and Li et al.(2006) Nat. Biotechnol. 24:210 (both describing antibody production inglycoengineered Pichia pastoris.); Mossner et al. (2010) Blood 115:4393;Shields et al. (2002) J. Biol. Chem. 277:26733; Shinkawa et al. (2003)J. Biol. Chem. 278:3466; EP 1176195B1. ADCC can also be enhanced asdescribed in PCT Publication WO 03/035835, which discloses use of avariant CHO cell line, Lec13, with reduced ability to attach fucose toAsn(297)-linked carbohydrates, also resulting in hypofucosylation ofantibodies expressed in that host cell (see also Shields, R. L. et al.(2002) J. Biol. Chem. 277:26733-26740). Alternatively, fucose analogsmay be added to culture medium during antibody production to inhibitincorporation of fucose into the carbohydrate on the antibody. WO2009/135181.

Increasing bisecting GlcNac structures in antibody-linkedoligosaccharides also enhances ADCC. PCT Publication WO 99/54342 byUmana et al. describes cell lines engineered to expressglycoprotein-modifying glycosyl transferases (e.g.,beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such thatantibodies expressed in the engineered cell lines exhibit increasedbisecting GlcNac structures which results in increased ADCC activity ofthe antibodies (see also Umana et al. (1999) Nat. Biotech. 17:176-180).

Additional glycosylation variants have been developed that are devoid ofgalactose, sialic acid, fucose and xylose residues (so-called GNGNglycoforms), which exhibit enhanced ADCC and ADCP but decreased CDC, aswell as others that are devoid of sialic acid, fucose and xylose(so-called G1/G2 glycoforms), which exhibit enhanced ADCC, ADCP and CDC.U.S. Pat. App. Pub. No. 2013/0149300. Antibodies having theseglycosylation patterns are optionally produced in genetically modifiedN. benthamiana plants in which the endogenous xylosyl and fucosyltransferase genes have been knocked-out.

Glycoengineering can also be used to modify the anti-inflammatoryproperties of an IgG construct by changing the α2,6 sialyl content ofthe carbohydrate chains attached at Asn297 of the Fc regions, wherein anincreased proportion of α2,6 sialylated forms results in enhancedanti-inflammatory effects. See Nimmerjahn et al. (2008) Ann. Rev.Immunol. 26:513. Conversely, reduction in the proportion of antibodieshaving α2,6 sialylated carbohydrates may be useful in cases whereanti-inflammatory properties are not wanted. Methods of modifying α2,6sialylation content of antibodies, for example by selective purificationof α2,6 sialylated forms or by enzymatic modification, are provided atU.S. Pat. Appl. Pub. No. 2008/0206246. In other embodiments, the aminoacid sequence of the Fc region may be modified to mimic the effect ofα2,6 sialylation, for example by inclusion of an F241A modification. WO2013/095966.

VIII. Antibody Physical Properties

Antibodies described herein can contain one or more glycosylation sitesin either the light or heavy chain variable region. Such glycosylationsites may result in increased immunogenicity of the antibody or analteration of the pK of the antibody due to altered antigen binding(Marshall et al (1972) Annu Rev Biochem 41:673-702; Gala and Morrison(2004) J. Immunol 172:5489-94; Wallick et al (1988) J Exp Med168:1099-109; Spiro (2002) Glycobiology 12:43R-56R; Parekh et al (1985)Nature 316:452-7; Mimura et al. (2000) Mol Immunol 37:697-706).Glycosylation has been known to occur at motifs containing an N-X-S/Tsequence. In some instances, it is preferred to have an anti-CD73antibody that does not contain variable region glycosylation. This canbe achieved either by selecting antibodies that do not contain theglycosylation motif in the variable region or by mutating residueswithin the glycosylation region.

In certain embodiments, the antibodies described herein do not containasparagine isomerism sites. The deamidation of asparagine may occur onN-G or D-G sequences and may result in the creation of an isoasparticacid residue that may introduce a kink into the polypeptide chain andmay decrease its stability (isoaspartic acid effect). For instance, ifthe amino acid sequence Asp-Gly is present in the heavy and/or lightchain CDR sequences of the antibody, the sequence is substituted with anamino acid sequence that does not undergo isomerization. In oneembodiment, the antibody comprises the heavy chain variable region CDR2sequence set forth in SEQ ID NO: 6, but wherein the Asp or Gly in theAsp-Gly sequence (VILYDGSNKYYPDSVKG; SEQ ID NO: 6) is replaced with anamino acid sequence that does not undergo isomerization, for example, anAsp-Ser or a Ser-Gly sequence.

Each antibody will have a unique isoelectric point (pI), which generallyfalls in the pH range between 6 and 9.5. The pI for an IgG1 antibodytypically falls within the pH range of 7-9.5 and the pI for an IgG4antibody typically falls within the pH range of 6-8. There isspeculation that antibodies with a pI outside the normal range may havesome unfolding and instability under in vivo conditions. Thus, it ispreferred to have an anti-CD73 antibody that contains a pI value thatfalls in the normal range. This can be achieved either by selectingantibodies with a pI in the normal range or by mutating charged surfaceresidues.

Each antibody will have a characteristic melting temperature, with ahigher melting temperature indicating greater overall stability in vivo(Krishnamurthy R and Manning M C (2002) Curr Pharm Biotechnol 3:361-71).Generally, it is preferred that the T_(M1) (the temperature of initialunfolding) be greater than 60° C., preferably greater than 65° C., evenmore preferably greater than 70° C. The melting point of an antibody canbe measured using differential scanning calorimetry (Chen et al (2003)Pharm Res 20:1952-60; Ghirlando et al (1999) Immunol Lett 68:47-52) orcircular dichroism (Murray et al. (2002) J. Chromatogr Sci 40:343-9). Ina preferred embodiment, antibodies are selected that do not degraderapidly. Degradation of an antibody can be measured using capillaryelectrophoresis (CE) and MALDI-MS (Alexander A J and Hughes D E (1995)Anal Chem 67:3626-32).

In another preferred embodiment, antibodies are selected that haveminimal aggregation effects, which can lead to the triggering of anunwanted immune response and/or altered or unfavorable pharmacokineticproperties. Generally, antibodies are acceptable with aggregation of 25%or less, preferably 20% or less, even more preferably 15% or less, evenmore preferably 10% or less and even more preferably 5% or less.Aggregation can be measured by several techniques, includingsize-exclusion column (SEC), high performance liquid chromatography(HPLC), and light scattering.

IX. Methods of Engineering Antibodies

As discussed above, the anti-CD73 antibodies having V_(H) and V_(L)sequences disclosed herein can be used to create new anti-CD73antibodies by modifying the VH and/or VL sequences, or the constantregion(s) attached thereto. Thus, in another aspect described herein,the structural features of an anti-CD73 antibody described herein, e.g.CD73.4, 11F11, 4C3, 4D4, 10D2, 11A6, 24H2, 5F8, 6E11 and/or 7A11, areused to create structurally related anti-CD73 antibodies that retain atleast one functional property of the antibodies described herein, suchas binding to human CD73 and cynomolgus CD73. For example, one or moreCDR regions of 11F11, 4C3, 4D4, 10D2, 11A6, 24H2, 5F8, 6E11 and/or 7A11,or mutations thereof, can be combined recombinantly with known frameworkregions and/or other CDRs to create additional,recombinantly-engineered, anti-CD73 antibodies described herein, asdiscussed above. Other types of modifications include those described inthe previous section. The starting material for the engineering methodis one or more of the V_(H) and/or V_(L) sequences provided herein, orone or more CDR regions thereof. To create the engineered antibody, itis not necessary to actually prepare (i.e., express as a protein) anantibody having one or more of the V_(H) and/or V_(L) sequences providedherein, or one or more CDR regions thereof. Rather, the informationcontained in the sequence(s) is used as the starting material to createa “second generation” sequence(s) derived from the original sequence(s)and then the “second generation” sequence(s) is prepared and expressedas a protein.

Accordingly, provided herein are methods for preparing an anti-CD73antibody comprising:

(a) providing: (i) a heavy chain variable region antibody sequencecomprising a CDR1 sequence selected from the group consisting of SEQ IDNOs: 5, 17, 33, 41, 53, 61, 69, 81, and 89, a CDR2 sequence selectedfrom the group consisting of SEQ ID NOs: 6, 18, 34, 42, 54, 62, 70, 82,and 90, and/or a CDR3 sequence selected from the group consisting of SEQID NOs: 7, 19, 35, 43, 55, 63, 71, 83, and 91; and (ii) a light chainvariable region antibody sequence comprising a CDR1 sequence selectedfrom the group consisting of SEQ ID NOs: 9, 13, 21, 25, 29, 37, 45, 49,57, 65, 73, 77, 85, and 93, a CDR2 sequence selected from the groupconsisting of SEQ ID NOs: 10, 14, 22, 26, 30, 38, 46, 50, 58, 66, 74,78, 86, and 94, and/or a CDR3 sequence selected from the groupconsisting of SEQ ID NOs: 11, 15, 23, 27, 31, 39, 47, 51, 59, 67, 75,79, 87, and 95;

(b) altering at least one amino acid residue within the heavy chainvariable region antibody sequence and/or the light chain variable regionantibody sequence to create at least one altered antibody sequence; and

(c) expressing the altered antibody sequence as a protein.

Standard molecular biology techniques can be used to prepare and expressthe altered antibody sequence.

Preferably, the antibody encoded by the altered antibody sequence(s) isone that retains one, some or all of the functional properties of theanti-CD73 antibodies described herein, which include those listed inTable 3.

The altered antibody may exhibit one or more, two or more, three ormore, four or more, five or more, six or more, seven or more, eight ormore, nine or more, ten, or all of the functional properties using thefunctional assays described herein. The functional properties of thealtered antibodies can be assessed using standard assays available inthe art and/or described herein, such as those set forth in the Examples(e.g., ELISAs, FACS).

In certain embodiments of the methods of engineering antibodiesdescribed herein, mutations can be introduced randomly or selectivelyalong all or part of an anti-CD73 antibody coding sequence and theresulting modified anti-CD73 antibodies can be screened for bindingactivity and/or other functional properties as described herein.Mutational methods have been described in the art. For example, PCTPublication WO 02/092780 by Short describes methods for creating andscreening antibody mutations using saturation mutagenesis, syntheticligation assembly, or a combination thereof. Alternatively, PCTPublication WO 03/074679 by Lazar et al. describes methods of usingcomputational screening methods to optimize physiochemical properties ofantibodies.

X. Nucleic Acid Molecules

Another aspect described herein pertains to nucleic acid molecules thatencode the antibodies described herein. The nucleic acids may be presentin whole cells, in a cell lysate, or in a partially purified orsubstantially pure form. A nucleic acid is “isolated” or “renderedsubstantially pure” when purified away from other cellular components orother contaminants, e.g., other cellular nucleic acids (e.g., otherchromosomal DNA, e.g., the chromosomal DNA that is linked to theisolated DNA in nature) or proteins, by standard techniques, includingalkaline/SDS treatment, CsCl banding, column chromatography, restrictionenzymes, agarose gel electrophoresis and others well known in the art.See, F. Ausubel, et al., ed. (1987) Current Protocols in MolecularBiology, Greene Publishing and Wiley Interscience, New York. A nucleicacid described herein can be, for example, DNA or RNA and may or may notcontain intronic sequences. In a certain embodiments, the nucleic acidis a cDNA molecule.

Nucleic acids described herein can be obtained using standard molecularbiology techniques. For antibodies expressed by hybridomas (e.g.,hybridomas prepared from transgenic mice carrying human immunoglobulingenes as described further below), cDNAs encoding the light and heavychains of the antibody made by the hybridoma can be obtained by standardPCR amplification or cDNA cloning techniques. For antibodies obtainedfrom an immunoglobulin gene library (e.g., using phage displaytechniques), nucleic acid encoding the antibody can be recovered fromthe library.

Preferred nucleic acids molecules described herein are those encodingthe VH and VL sequences of the anti-CD73 antibodies described herein,e.g., CD73.4 11F11-1, 11F11-2, 4C3-1, 4C3-2, 4C3-3, 4D4, 10D2-1, 10D2-2,11A6, 24H2, 5F8-1, 5F8-2, 6E11, 7A11, CD73.3 and/or CD73.4 monoclonalantibodies. DNA sequences encoding the VH sequences of CD73.4 (CD73.4-1and CD73.4-2) 11F11 (11F11-1 and 11F11-2), 4C3 (4C3-1, 4C3-2 and 4C3-3),4D4, 10D2 (10D2-1 and 10D2-2), 11A6, 24H2, 5F8 (5F8-1 and 5F8-2), 6E11,7A11, CD73.3 and CD73.4 are set forth in SEQ ID NOs: 4, 16, 32, 40, 52,60, 68, 80, 88, 135, and 170, respectively. DNA sequences encoding theVL sequences of 11F11-1, 11F11-2, 4C3-1, 4C3-2, 4C3-3, 4D4, 10D2-1,10D2-2, 11A6, 24H2, 5F8-1, 5F8-2, 6E17A11, CD73.3 and/or CD73.4 are setforth in SEQ ID NOs: 8, 12, 20, 24, 28, 36, 44, 48, 56, 64, 72, 76, 84,and 92, respectively.

Once DNA fragments encoding VH and VL segments are obtained, these DNAfragments can be further manipulated by standard recombinant DNAtechniques, for example to convert the variable region genes tofull-length antibody chain genes, to Fab fragment genes or to a scFvgene. In these manipulations, a VL- or VH-encoding DNA fragment isoperatively linked to another DNA fragment encoding another protein,such as an antibody constant region or a flexible linker. The term“operatively linked”, as used in this context, is intended to mean thatthe two DNA fragments are joined such that the amino acid sequencesencoded by the two DNA fragments remain in-frame.

The isolated DNA encoding the VH region can be converted to afull-length heavy chain gene by operatively linking the VH-encoding DNAto another DNA molecule encoding heavy chain constant regions (hinge,CH1, CH2 and/or CH3). The sequences of human heavy chain constant regiongenes are known in the art (see e.g., Kabat, E. A., et al. (1991)Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242)and DNA fragments encompassing these regions can be obtained by standardPCR amplification. The heavy chain constant region can be an IgG1, IgG2,IgG3, IgG4, IgA, IgE, IgM or IgD constant region, for example, an IgG1region. For a Fab fragment heavy chain gene, the VH-encoding DNA can beoperatively linked to another DNA molecule encoding only the heavy chainCH1 constant region.

The isolated DNA encoding the VL region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the VL-encoding DNA to another DNA molecule encodingthe light chain constant region, CL. The sequences of human light chainconstant region genes are known in the art (see e.g., Kabat, E. A., etal. (1991) Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242) and DNA fragments encompassing these regions can beobtained by standard PCR amplification. The light chain constant regioncan be a kappa or lambda constant region.

To create a scFv gene, the VH- and VL-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly₄-Ser)₃, such that the VH and VLsequences can be expressed as a contiguous single-chain protein, withthe VL and VH regions joined by the flexible linker (see e.g., Bird etal. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad.Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).

Also provided herein are nucleic acid molecules encoding VH and VLsequences or full length heavy and light chains that are homologous tothose of antibodies described herein, e.g., the 11F11-1, 11F11-2, 4C3-1,4C3-2, 4C3-3, 4D4, 10D2-1, 10D2-2, 11A6, 24H2, 5F8-1, 5F8-2, 6E17A11,CD73.3 and/or CD73.4 monoclonal antibodies. Exemplary nucleic acidmolecules encode VH and VL sequences that are at least 70% identical,for example, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or at least 99% identical, to nucleic acid molecules encodingthe VH and VL sequences or the full length heavy and light chains of the11F11-1, 11F11-2, 4C3-1, 4C3-2, 4C3-3, 4D4, 10D2-1, 10D2-2, 11A6, 24H2,5F8-1, 5F8-2, 6E11 7A11, CD73.3 and/or CD73.4 monoclonal antibodies,e.g., the sequences set forth in Table 35. For example, provided hereinare anti-CD73 antibodies comprising a VH chain and a VL chain that areencoded by nucleotides sequences that are at least 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 139 and SEQ ID NO:140 or 141; SEQ ID NO: 237 and SEQ ID NO: 140 or 141; SEQ ID NO: 142 andSEQ ID NO: 143, 144 or 145; SEQ ID NO: 146 and SEQ ID NO: 147; SEQ IDNO: 148 and SEQ ID NO: 149 or 150; SEQ ID NO: 151 and SEQ ID NO: 152;SEQ ID NO: 153 and SEQ ID NO: 154; SEQ ID NO: 155 and SEQ ID NO: 156 or157 or 242; SEQ ID NO: 158 and SEQ ID NO: 159; SEQ ID NO: 160 and SEQ IDNO: 161. Also provided are anti-CD73 antibodies comprising a heavy chainand a light chain that are encoded by nucleotides sequences that are atleast 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ IDNOs: 134, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,226, 227, 228, 229, 230, 231, 232, 233, 234, 243, 266 (heavy chain) andSEQ ID NO: 244 or 245 (light chain); SEQ ID NO: 211, 212, 213 or 246 andSEQ ID NO: 247, 248 or 249; SEQ ID NO: 235, 236 or 250 and 251; SEQ IDNO: 252 and SEQ ID NO: 253 or 254; SEQ ID NO: 255 and SEQ ID NO: 256;SEQ ID NO: 257 and SEQ ID NO: 258; SEQ ID NO: 259 and SEQ ID NO: 260 or261; SEQ ID NO: 262 and SEQ ID NO: 263; SEQ ID NO: 264 and SEQ ID NO:265. Also provided herein are nucleic acid molecules with silentmutations (i.e., base changes that do not alter the resulting amino acidsequence upon translation of nucleic acid molecule), e.g., for codonoptimization.

XI. Antibody Generation

Various antibodies of the present invention, e.g. those that competewith or bind to the same epitope as the anti-human CD73 antibodiesdisclosed herein, can be produced using a variety of known techniques,such as the standard somatic cell hybridization technique described byKohler and Milstein, Nature 256: 495 (1975). Although somatic cellhybridization procedures are preferred, in principle, other techniquesfor producing monoclonal antibodies also can be employed, e.g., viral oroncogenic transformation of B lymphocytes, phage display technique usinglibraries of human antibody genes.

The preferred animal system for preparing hybridomas is the murinesystem. Hybridoma production in the mouse is a very well-establishedprocedure. Immunization protocols and techniques for isolation ofimmunized splenocytes for fusion are known in the art. Fusion partners(e.g., murine myeloma cells) and fusion procedures are also known.

Chimeric or humanized antibodies described herein can be prepared basedon the sequence of a murine monoclonal antibody prepared as describedabove. DNA encoding the heavy and light chain immunoglobulins can beobtained from the murine hybridoma of interest and engineered to containnon-murine (e.g., human) immunoglobulin sequences using standardmolecular biology techniques. For example, to create a chimericantibody, the murine variable regions can be linked to human constantregions using methods known in the art (see e.g., U.S. Pat. No.4,816,567 to Cabilly et al.). To create a humanized antibody, the murineCDR regions can be inserted into a human framework using methods knownin the art (see e.g., U.S. Pat. No. 5,225,539 to Winter, and U.S. Pat.Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.).

In one embodiment, the antibodies described herein are human monoclonalantibodies. Such human monoclonal antibodies directed against CD73 canbe generated using transgenic or transchromosomic mice carrying parts ofthe human immune system rather than the mouse system. These transgenicand transchromosomic mice include mice referred to herein as HuMAb miceand KM mice, respectively, and are collectively referred to herein as“human Ig mice.”

The HuMAb Mouse® (Medarex, Inc.) contains human immunoglobulin geneminiloci that encode unrearranged human heavy (μ and γ) and κ lightchain immunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (see e.g., Lonberg, et al.(1994) Nature 368(6474): 856-859). Accordingly, the mice exhibit reducedexpression of mouse IgM or κ, and in response to immunization, theintroduced human heavy and light chain transgenes undergo classswitching and somatic mutation to generate high affinity human IgGκmonoclonal (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N.(1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. andHuszar, D. (1995) Intern. Rev. Immunol. 13: 65-93, and Harding, F. andLonberg, N. (1995) Ann. N. Y. Acad. Sci. 764:536-546). The preparationand use of HuMab mice, and the genomic modifications carried by suchmice, is further described in Taylor, L. et al. (1992) Nucleic AcidsResearch 20:6287-6295; Chen, J. et al. (1993) International Immunology5: 647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA90:3720-3724; Choi et al. (1993) Nature Genetics 4:117-123; Chen, J. etal. (1993) EMBO J. 12: 821-830; Tuaillon et al. (1994) J. Immunol.152:2912-2920; Taylor, L. et al. (1994) International Immunology 6:579-591; and Fishwild, D. et al. (1996) Nature Biotechnology 14:845-851, the contents of all of which are hereby specificallyincorporated by reference in their entirety. See further, U.S. Pat. Nos.5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397;5,661,016; 5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay;U.S. Pat. No. 5,545,807 to Surani et al.; PCT Publication Nos. WO92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO 98/24884 and WO99/45962, all to Lonberg and Kay; and PCT Publication No. WO 01/14424 toKorman et al.

In certain embodiments, antibodies described herein are raised using amouse that carries human immunoglobulin sequences on transgenes andtranschromosomes, such as a mouse that carries a human heavy chaintransgene and a human light chain transchromosome. Such mice, referredto herein as “KM mice”, are described in detail in PCT Publication WO02/43478 to Ishida et al.

Still further, alternative transgenic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-CD73 antibodies described herein. For example, an alternativetransgenic system referred to as the Xenomouse (Abgenix, Inc.) can beused; such mice are described in, for example, U.S. Pat. Nos. 5,939,598;6,075,181; 6,114,598; 6, 150,584 and 6,162,963 to Kucherlapati et al.

Moreover, alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-CD73 antibodies described herein. For example, mice carrying both ahuman heavy chain transchromosome and a human light chaintranschromosome, referred to as “TC mice” can be used; such mice aredescribed in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA97:722-727. Furthermore, cows carrying human heavy and light chaintranschromosomes have been described in the art (Kuroiwa et al. (2002)Nature Biotechnology 20:889-894) and can be used to raise anti-CD73antibodies described herein.

Additional mouse systems described in the art for raising humanantibodies, e.g., human anti-CD73 antibodies, include (i) theVelocImmune® mouse (Regeneron Pharmaceuticals, Inc.), in which theendogenous mouse heavy and light chain variable regions have beenreplaced, via homologous recombination, with human heavy and light chainvariable regions, operatively linked to the endogenous mouse constantregions, such that chimeric antibodies (human V/mouse C) are raised inthe mice, and then subsequently converted to fully human antibodiesusing standard recombinant DNA techniques; and (ii) the MeMo® mouse(Merus Biopharmaceuticals, Inc.), in which the mouse containsunrearranged human heavy chain variable regions but a single rearrangedhuman common light chain variable region. Such mice, and use thereof toraise antibodies, are described in, for example, WO 2009/15777, US2010/0069614, WO 2011/072204, WO 2011/097603, WO 2011/163311, WO2011/163314, WO 2012/148873, US 2012/0070861 and US 2012/0073004.

Human monoclonal antibodies described herein can also be prepared usingphage display methods for screening libraries of human immunoglobulingenes. Such phage display methods for isolating human antibodies areestablished in the art. See for example: U.S. Pat. Nos. 5,223,409;5,403,484; and U.S. Pat. No. 5,571,698 to Ladner et al.; U.S. Pat. Nos.5,427,908 and 5,580,717 to Dower et al.; U.S. Pat. Nos. 5,969,108 and6,172,197 to McCafferty et al.; and U.S. Pat. Nos. 5,885,793; 6,521,404;6,544,731; 6,555,313; 6,582,915 and 6,593,081 to Griffiths et al.

Human monoclonal antibodies described herein can also be prepared usingSCID mice into which human immune cells have been reconstituted suchthat a human antibody response can be generated upon immunization. Suchmice are described in, for example, U.S. Pat. Nos. 5,476,996 and5,698,767 to Wilson et al.

Immunizations

To generate fully human antibodies to CD73, transgenic ortranschromosomal mice containing human immunoglobulin genes (e.g.,HCo12, HCo7 or KM mice) can be immunized with a purified or enrichedpreparation of the CD73 antigen and/or cells expressing CD73, asdescribed for other antigens, for example, by Lonberg et al. (1994)Nature 368(6474): 856-859; Fishwild et al. (1996) Nature Biotechnology14: 845-851 and WO 98/24884. Alternatively, mice can be immunized withDNA encoding human CD73. Preferably, the mice will be 6-16 weeks of ageupon the first infusion. For example, a purified or enriched preparation(5-50 μg) of the recombinant CD73 antigen can be used to immunize theHuMAb mice intraperitoneally. In the event that immunizations using apurified or enriched preparation of the CD73antigen do not result inantibodies, mice can also be immunized with cells expressing CD73, e.g.,a cell line, to promote immune responses. Exemplary cell lines includeCD73-overexpressing stable CHO and Raji cell lines.

Cumulative experience with various antigens has shown that the HuMAbtransgenic mice respond best when initially immunized intraperitoneally(IP) or subcutaneously (SC) with antigen in Ribi's adjuvant, followed byevery other week IP/SC immunizations (up to a total of 10) with antigenin Ribi's adjuvant. The immune response can be monitored over the courseof the immunization protocol with plasma samples being obtained byretroorbital bleeds. The plasma can be screened by ELISA and FACS (asdescribed below), and mice with sufficient titers of anti-CD73 humanimmunoglobulin can be used for fusions. Mice can be boostedintravenously with antigen 3 days before sacrifice and removal of thespleen and lymph nodes. It is expected that 2-3 fusions for eachimmunization may need to be performed. Between 6 and 24 mice aretypically immunized for each antigen. Usually, HCo7, HCo12, and KMstrains are used. In addition, both HCo7 and HCo12 transgene can be bredtogether into a single mouse having two different human heavy chaintransgenes (HCo7/HCo12).

Generation of Hybridomas Producing Monoclonal Antibodies to CD73

To generate hybridomas producing human monoclonal antibodies describedherein, splenocytes and/or lymph node cells from immunized mice can beisolated and fused to an appropriate immortalized cell line, such as amouse myeloma cell line. The resulting hybridomas can be screened forthe production of antigen-specific antibodies. For example, single cellsuspensions of splenic lymphocytes from immunized mice can be fused toSp2/0 nonsecreting mouse myeloma cells (ATCC, CRL 1581) with 50% PEG.Cells are plated at approximately 2×10⁵ in flat bottom microtiter plate,followed by a two week incubation in selective medium containing 10%fetal Clone Serum, 18% “653” conditioned media, 5% origen (IGEN), 4 mML-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055 mM2-mercaptoethanol, 50 units/ml penicillin, 50 mg/ml streptomycin, 50mg/ml gentamycin and 1×HAT (Sigma). After approximately two weeks, cellscan be cultured in medium in which the HAT is replaced with HT.Individual wells can then be screened by ELISA for human monoclonal IgMand IgG antibodies. Once extensive hybridoma growth occurs, medium canbe observed usually after 10-14 days. The antibody secreting hybridomascan be replated, screened again, and if still positive for human IgG,the monoclonal antibodies can be subcloned at least twice by limitingdilution. The stable subclones can then be cultured in vitro to generatesmall amounts of antibody in tissue culture medium for characterization.

To purify human monoclonal antibodies, selected hybridomas can be grownin two-liter spinner-flasks for monoclonal antibody purification.Supernatants can be filtered and concentrated before affinitychromatography with protein A-sepharose (Pharmacia, Piscataway, N.J.).Eluted IgG can be checked by gel electrophoresis and high performanceliquid chromatography to ensure purity. The buffer solution can beexchanged into PBS, and the concentration can be determined by OD280using 1.43 extinction coefficient. The monoclonal antibodies can bealiquoted and stored at −80° C.

XII. Antibody Manufacture Generation of Transfectomas ProducingMonoclonal Antibodies to CD73

Antibodies of the present invention, including both specific antibodiesfor which sequences are provided and other, related anti-CD73antibodies, can be produced in a host cell transfectoma using, forexample, a combination of recombinant DNA techniques and genetransfection methods as is well known in the art (Morrison, S. (1985)Science 229:1202).

For example, to express antibodies, or antibody fragments thereof, DNAsencoding partial or full-length light and heavy chains, can be obtainedby standard molecular biology techniques (e.g., PCR amplification orcDNA cloning using a hybridoma that expresses the antibody of interest)and the DNAs can be inserted into expression vectors such that the genesare operatively linked to transcriptional and translational controlsequences. In this context, the term “operatively linked” is intended tomean that an antibody gene is ligated into a vector such thattranscriptional and translational control sequences within the vectorserve their intended function of regulating the transcription andtranslation of the antibody gene. The expression vector and expressioncontrol sequences are chosen to be compatible with the expression hostcell used. The antibody light chain gene and the antibody heavy chaingene can be inserted into separate vector or both genes are insertedinto the same expression vector. The antibody genes are inserted intothe expression vector(s) by standard methods (e.g., ligation ofcomplementary restriction sites on the antibody gene fragment andvector, or blunt end ligation if no restriction sites are present). Thelight and heavy chain variable regions of the antibodies describedherein can be used to create full-length antibody genes of any antibodyisotype by inserting them into expression vectors already encoding heavychain constant and light chain constant regions of the desired isotypesuch that the V_(H) segment is operatively linked to the CH segment(s)within the vector and the V_(L) segment is operatively linked to the CLsegment within the vector. Additionally or alternatively, therecombinant expression vector can encode a signal peptide thatfacilitates secretion of the antibody chain from a host cell. Theantibody chain gene can be cloned into the vector such that the signalpeptide is linked in-frame to the amino terminus of the antibody chaingene. The signal peptide can be an immunoglobulin signal peptide or aheterologous signal peptide (i.e., a signal peptide from anon-immunoglobulin protein).

In addition to the antibody chain genes, recombinant expression vectorsmay carry regulatory sequences that control the expression of theantibody chain genes in a host cell. The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals) that control the transcriptionor translation of the antibody chain genes. Such regulatory sequencesare described, for example, in Goeddel (Gene Expression Technology.Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). Itwill be appreciated by those skilled in the art that the design of theexpression vector, including the selection of regulatory sequences, maydepend on such factors as the choice of the host cell to be transformed,the level of expression of protein desired, etc. Preferred regulatorysequences for mammalian host cell expression include viral elements thatdirect high levels of protein expression in mammalian cells, such aspromoters and/or enhancers derived from cytomegalovirus (CMV), SimianVirus 40 (SV40), adenovirus, (e.g., the adenovirus major late promoter(AdMLP) and polyoma. Alternatively, nonviral regulatory sequences may beused, such as the ubiquitin promoter or β-globin promoter. Stillfurther, regulatory elements composed of sequences from differentsources, such as the SRα promoter system, which contains sequences fromthe SV40 early promoter and the long terminal repeat of human T cellleukemia virus type 1 (Takebe, Y. et al. (1988) Mol. Cell. Biol.8:466-472).

In addition to the antibody chain genes and regulatory sequences,recombinant expression vectors may carry additional sequences, such assequences that regulate replication of the vector in host cells (e.g.,origins of replication) and selectable marker genes. The selectablemarker gene facilitates selection of host cells into which the vectorhas been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and5,179,017, all by Axel et al.). For example, typically the selectablemarker gene confers resistance to drugs, such as G418, hygromycin ormethotrexate, on a host cell into which the vector has been introduced.Preferred selectable marker genes include the dihydrofolate reductase(DHFR) gene (for use in dhfr-host cells with methotrexateselection/amplification) and the neo gene (for G418 selection).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell bystandard techniques. The various forms of the term “transfection” areintended to encompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. Although it is theoreticallypossible to express the antibodies described herein in eitherprokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells, and most preferably mammalian host cells, is the mostpreferred because such eukaryotic cells, and in particular mammaliancells, are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active antibody. Prokaryoticexpression of antibody genes has been reported to be ineffective forproduction of high yields of active antibody (Boss, M. A. and Wood, C.R. (1985) Immunology Today 6:12-13). Antibodies of the present inventioncan also be produced in glycoengineered strains of the yeast Pichiapastoris. Li et al. (2006) Nat. Biotechnol. 24:210.

Preferred mammalian host cells for expressing the recombinant antibodiesdescribed herein include Chinese Hamster Ovary (CHO cells) (includingdhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad.Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., asdescribed in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol.159:601-621), NSO myeloma cells, COS cells and SP2 cells. In particular,for use with NSO myeloma cells, another preferred expression system isthe GS gene expression system disclosed in WO 87/04462, WO 89/01036 andEP 338,841. When recombinant expression vectors encoding antibody genesare introduced into mammalian host cells, the antibodies are produced byculturing the host cells for a period of time sufficient to allow forexpression of the antibody in the host cells or, more preferably,secretion of the antibody into the culture medium in which the hostcells are grown. Antibodies can be recovered from the culture mediumusing standard protein purification methods.

The N- and C-termini of antibody polypeptide chains of the presentinvention may differ from the expected sequence due to commonly observedpost-translational modifications. For example, C-terminal lysineresidues are often missing from antibody heavy chains. Dick et al.(2008) Biotechnol. Bioeng. 100:1132. N-terminal glutamine residues, andto a lesser extent glutamate residues, are frequently converted topyroglutamate residues on both light and heavy chains of therapeuticantibodies. Dick et al. (2007) Biotechnol. Bioeng. 97:544; Liu et al.(2011) JBC 28611211; Liu et al. (2011) J. Biol. Chem. 286:11211.

XIII. Assays

Antibodies described herein can be tested for binding to CD73 by, forexample, standard ELISA. Briefly, microtiter plates are coated withpurified CD73 at 1-2 μg/ml in PBS, and then blocked with 5% bovine serumalbumin in PBS. Dilutions of antibody (e.g., dilutions of plasma fromCD73-immunized mice) are added to each well and incubated for 1-2 hoursat 37° C. The plates are washed with PBS/Tween and then incubated withsecondary reagent (e.g., for human antibodies, a goat-anti-human IgGFc-specific polyclonal reagent) conjugated to horseradish peroxidase(HRP) for 1 hour at 37° C. After washing, the plates are developed withABTS substrate (Moss Inc, product: ABTS-1000) and analyzed by aspectrophotometer at OD 415-495. Sera from immunized mice are thenfurther screened by flow cytometry for binding to a cell line expressinghuman CD73, but not to a control cell line that does not express CD73.Briefly, the binding of anti-CD73 antibodies is assessed by incubatingCD73 expressing CHO cells with the anti-CD73 antibody at 1:20 dilution.The cells are washed and binding is detected with a PE-labeledanti-human IgG Ab. Flow cytometric analyses are performed using aFACScan flow cytometry (Becton Dickinson, San Jose, Calif.). Preferably,mice which develop the highest titers will be used for fusions.

An ELISA assay as described above can be used to screen for antibodiesand, thus, hybridomas that produce antibodies that show positivereactivity with the CD73 immunogen. Hybridomas that produce antibodiesthat bind, preferably with high affinity, to CD73 can then be subclonedand further characterized. One clone from each hybridoma, which retainsthe reactivity of the parent cells (by ELISA), can then be chosen formaking a cell bank, and for antibody purification.

To purify anti-CD73 antibodies, selected hybridomas can be grown intwo-liter spinner-flasks for monoclonal antibody purification.Supernatants can be filtered and concentrated before affinitychromatography with protein A-sepharose (Pharmacia, Piscataway, N.J.).Eluted IgG can be checked by gel electrophoresis and high performanceliquid chromatography to ensure purity. The buffer solution can beexchanged into PBS, and the concentration can be determined by OD₂₈₀using 1.43 extinction coefficient. The monoclonal antibodies can bealiquoted and stored at −80° C.

To determine if the selected anti-CD73 monoclonal antibodies bind tounique epitopes, each antibody can be biotinylated using commerciallyavailable reagents (Pierce, Rockford, Ill.). Biotinylated mAb bindingcan be detected with a streptavidin labeled probe. Competition studiesusing unlabeled monoclonal antibodies and biotinylated monoclonalantibodies can be performed using CD73 coated-ELISA plates as describedabove.

To determine the isotype of purified antibodies, isotype ELISAs can beperformed using reagents specific for antibodies of a particularisotype. For example, to determine the isotype of a human monoclonalantibody, wells of microtiter plates can be coated with 1 μg/ml ofanti-human immunoglobulin overnight at 4° C. After blocking with 1% BSA,the plates are reacted with 1 μg/ml or less of test monoclonalantibodies or purified isotype controls, at ambient temperature for oneto two hours. The wells can then be reacted with either human IgG1 orhuman IgM-specific alkaline phosphatase-conjugated probes. Plates aredeveloped and analyzed as described above.

To test the binding of monoclonal antibodies to live cells expressingCD73, flow cytometry can be used, as described in the Examples. Briefly,cell lines expressing membrane-bound CD73 (grown under standard growthconditions) are mixed with various concentrations of monoclonalantibodies in PBS containing 0.1% BSA at 4° C. for 1 hour. Afterwashing, the cells are reacted with Fluorescein-labeled anti-IgGantibody under the same conditions as the primary antibody staining. Thesamples can be analyzed by FACScan instrument using light and sidescatter properties to gate on single cells and binding of the labeledantibodies is determined. An alternative assay using fluorescencemicroscopy may be used (in addition to or instead of) the flow cytometryassay. Cells can be stained exactly as described above and examined byfluorescence microscopy. This method allows visualization of individualcells, but may have diminished sensitivity depending on the density ofthe antigen.

Anti-CD73 antibodies can be further tested for reactivity with the CD73antigen by Western blotting. Briefly, cell extracts from cellsexpressing CD73 can be prepared and subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis. After electrophoresis, the separatedantigens will be transferred to nitrocellulose membranes, blocked with20% mouse serum, and probed with the monoclonal antibodies to be tested.IgG binding can be detected using anti-IgG alkaline phosphatase anddeveloped with BCIP/NBT substrate tablets (Sigma Chem. Co., St. Louis,Mo.).

Methods for analyzing binding affinity, cross-reactivity, and bindingkinetics of various anti-CD73 antibodies include standard assays knownin the art, for example, BIACORE® surface plasmon resonance (SPR)analysis using a BIACORE® 2000 SPR instrument (Biacore AB, Uppsala,Sweden).

XIV. Immunoconjugates and Antibody Derivatives

Antibodies described herein can be used for diagnostic purposes,including sample testing and in vivo imaging, and for this purpose theantibody (or binding fragment thereof) can be conjugated to anappropriate detectable agent, to form an immunoconjugate. For diagnosticpurposes, appropriate agents are detectable labels that includeradioisotopes, for whole body imaging, and radioisotopes, enzymes,fluorescent labels and other suitable antibody tags for sample testing.

The detectable labels can be any of the various types used currently inthe field of in vitro diagnostics, including particulate labelsincluding metal sols such as colloidal gold, isotopes such as I¹²⁵ orTc⁹⁹ presented for instance with a peptidic chelating agent of the N₂S₂,N₃S or N₄ type, chromophores including fluorescent markers, biotin,luminescent markers, phosphorescent markers and the like, as well asenzyme labels that convert a given substrate to a detectable marker, andpolynucleotide tags that are revealed following amplification such as bypolymerase chain reaction. A biotinylated antibody would then bedetectable by avidin or streptavidin binding. Suitable enzyme labelsinclude horseradish peroxidase, alkaline phosphatase and the like. Forinstance, the label can be the enzyme alkaline phosphatase, detected bymeasuring the presence or formation of chemiluminescence followingconversion of 1,2 dioxetane substrates such as adamantyl methoxyphosphoryloxy phenyl dioxetane (AMPPD), disodium3-(4-(methoxyspiro{1,2-dioxetane-3,2′-(5′-chloro)tricyclo{3.3.1.13,7}decan}-4-yl) phenyl phosphate (CSPD), as well as CDP and CDP-star®or other luminescent substrates well-known to those in the art, forexample the chelates of suitable lanthanides such as Terbium(III) andEuropium(III). The detection means is determined by the chosen label.Appearance of the label or its reaction products can be achieved usingthe naked eye, in the case where the label is particulate andaccumulates at appropriate levels, or using instruments such as aspectrophotometer, a luminometer, a fluorimeter, and the like, all inaccordance with standard practice.

Preferably, conjugation methods result in linkages which aresubstantially (or nearly) non-immunogenic, e.g., peptide- (i.e. amide-),sulfide-, (sterically hindered), disulfide-, hydrazone-, and etherlinkages. These linkages are nearly non-immunogenic and show reasonablestability within serum (see e.g. Senter, P. D., Curr. Opin. Chem. Biol.13 (2009) 235-244; WO 2009/059278; WO 95/17886).

Depending on the biochemical nature of the moiety and the antibody,different conjugation strategies can be employed. In case the moiety isnaturally occurring or recombinant polypeptide of between 50 to 500amino acids, there are standard procedures in text books describing thechemistry for synthesis of protein conjugates, which can be easilyfollowed by the skilled artisan (see e.g. Hackenberger, C. P. R., andSchwarzer, D., Angew. Chem. Int. Ed. Engl. 47 (2008) 10030-10074). Inone embodiment the reaction of a maleinimido moiety with a cysteineresidue within the antibody or the moiety is used. This is an especiallysuitable coupling chemistry in case e.g. a Fab or Fab′-fragment of anantibody is used. Alternatively in one embodiment coupling to theC-terminal end of the antibody or moiety is performed. C-terminalmodification of a protein, e.g. of a Fab-fragment can be performed asdescribed (Sunbul, M. and Yin, J., Org. Biomol. Chem. 7 (2009)3361-3371).

In general site specific reaction and covalent coupling is based ontransforming a natural amino acid into an amino acid with a reactivitywhich is orthogonal to the reactivity of the other functional groupspresent. For example, a specific cysteine within a rare sequence contextcan be enzymatically converted in an aldehyde (see Frese, M. A., andDierks, T., ChemBioChem. 10 (2009) 425-427). It is also possible toobtain a desired amino acid modification by utilizing the specificenzymatic reactivity of certain enzymes with a natural amino acid in agiven sequence context (see, e.g., Taki, M. et al., Prot. Eng. Des. Sel.17 (2004) 119-126; Gautier, A. et al. Chem. Biol. 15 (2008) 128-136.Protease-catalyzed formation of C—N bonds is described at Bordusa, F.,Highlights in Bioorganic Chemistry (2004) 389-403.

Site specific reaction and covalent coupling can also be achieved by theselective reaction of terminal amino acids with appropriate modifyingreagents. The reactivity of an N-terminal cysteine with benzonitrils(see Ren, H. et al., Angew. Chem. Int. Ed. Engl. 48 (2009) 9658-9662)can be used to achieve a site-specific covalent coupling. Nativechemical ligation can also rely on C-terminal cysteine residues (Taylor,E. Vogel; Imperiali, B, Nucleic Acids and Molecular Biology (2009), 22(Protein Engineering), 65-96). EP 1 074 563 describes a conjugationmethod which is based on the faster reaction of a cysteine within astretch of negatively charged amino acids than a cysteine located in astretch of positively charged amino acids.

The moiety may also be a synthetic peptide or peptide mimic. In case apolypeptide is chemically synthesized, amino acids with orthogonalchemical reactivity can be incorporated during such synthesis (see e.g.de Graaf, A. J. et al., Bioconjug. Chem. 20 (2009) 1281-1295). Since agreat variety of orthogonal functional groups is at stake and can beintroduced into a synthetic peptide, conjugation of such peptide to alinker is standard chemistry.

In order to obtain a mono-labeled polypeptide the conjugate with 1:1stoichiometry may be separated by chromatography from other conjugationside-products. This procedure can be facilitated by using a dye labeledbinding pair member and a charged linker. By using this kind of labeledand highly negatively charged binding pair member, mono conjugatedpolypeptides are easily separated from non-labeled polypeptides andpolypeptides which carry more than one linker, since the difference incharge and molecular weight can be used for separation. The fluorescentdye can be useful for purifying the complex from un-bound components,like a labeled monovalent binder.

In one embodiment the moiety attached to an anti-CD73 antibody isselected from the group consisting of a binding moiety, a labelingmoiety, and a biologically active moiety.

Antibodies described herein may also be conjugated to a therapeuticagent to form an immunoconjugate such as an antibody-drug conjugate(ADC). Suitable therapeutic agents include antimetabolites, alkylatingagents, DNA minor groove binders, DNA intercalators, DNA crosslinkers,histone deacetylase inhibitors, nuclear export inhibitors, proteasomeinhibitors, topoisomerase I or II inhibitors, heat shock proteininhibitors, tyrosine kinase inhibitors, antibiotics, and anti-mitoticagents. In the ADC, the antibody and therapeutic agent preferably areconjugated via a linker cleavable such as a peptidyl, disulfide, orhydrazone linker. More preferably, the linker is a peptidyl linker suchas Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val (SEQ IDNO: 219), Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys,Cit, Ser, or Glu. The ADCs can be prepared as described in U.S. Pat.Nos. 7,087,600; 6,989,452; and 7,129,261; PCT Publications WO 02/096910;WO 07/038658; WO 07/051081; WO 07/059404; WO 08/083312; and WO08/103693; U.S. Patent Publications 20060024317; 20060004081; and20060247295; the disclosures of which are incorporated herein byreference. Other uses for anti-CD73 antibodies, e.g., as monotherapy,are provided elsewhere herein, e.g., in the section pertaining tocombination treatments.

More specifically, in an ADC, the antibody is conjugated to a drug, withthe antibody functioning as a targeting agent for directing the ADC to atarget cell expressing its antigen, such as a cancer cell. Preferably,the antigen is a tumor associated antigen, i.e., one that is uniquelyexpressed or overexpressed by the cancer cell. Once there, the drug isreleased, either inside the target cell or in its vicinity, to act as atherapeutic agent. For a review on the mechanism of action and use ofADCs in cancer therapy, see Schrama et al., Nature Rev. Drug Disc. 2006,5, 147.

For cancer treatment, the drug preferably is a cytotoxic drug thatcauses death of the targeted cancer cell. Cytotoxic drugs that can beused in ADCs include the following types of compounds and their analogsand derivatives:

-   (a) enediynes such as calicheamicin (see, e.g., Lee et al., J. Am.    Chem. Soc. 1987, 109, 3464 and 3466) and uncialamycin (see, e.g.,    Davies et al., WO 2007/038868 A2 (2007) and Chowdari et al., U.S.    Pat. No. 8,709,431 B2 (2012));-   (b) tubulysins (see, e.g., Domling et al., U.S. Pat. No. 7,778,814    B2 (2010); Cheng et al., U.S. Pat. No. 8,394,922 B2 (2013); and Cong    et al., US 2014/0227295 A1;-   (c) CC-1065 and duocarmycin (see, e.g., Boger, U.S. Pat. No.    6,5458,530 B1 (2003); Sufi et al., U.S. Pat. No. 8,461,117 B2    (2013); and Zhang et al., US 2012/0301490 A1 (2012));-   (d) epothilones (see, e.g., Vite et al., US 2007/0275904 A1 (2007)    and U.S. RE42930 E (2011));-   (e) auristatins (see, e.g., Senter et al., U.S. Pat. No. 6,844,869    B2 (2005) and Doronina et al., U.S. Pat. No. 7,498,298 B2 (2009));-   (f) pyrrolobezodiazepine (PBD) dimers (see, e.g., Howard et al., US    2013/0059800 A1(2013); US 2013/0028919 A1 (2013); and WO 2013/041606    A1 (2013)); and-   (g) maytansinoids such as DM1 and DM4 (see, e.g., Chari et al., U.S.    Pat. No. 5,208,020 (1993) and Amphlett et al., U.S. Pat. No.    7,374,762 B2 (2008)).

XV. Bispecific Molecules

Antibodies described herein may be used for forming bispecificmolecules. An anti-CD73 antibody, or antigen-binding portions thereof,can be derivatized or linked to another functional molecule, e.g.,another peptide or protein (e.g., another antibody or ligand for areceptor) to generate a bispecific molecule that binds to at least twodifferent binding sites or target molecules. The antibody describedherein may in fact be derivatized or linked to more than one otherfunctional molecule to generate multispecific molecules that bind tomore than two different binding sites and/or target molecules; suchmultispecific molecules are also intended to be encompassed by the term“bispecific molecule” as used herein. To create a bispecific moleculedescribed herein, an antibody described herein can be functionallylinked (e.g., by chemical coupling, genetic fusion, noncovalentassociation or otherwise) to one or more other binding molecules, suchas another antibody, antibody fragment, peptide or binding mimetic, suchthat a bispecific molecule results.

Accordingly, provided herein are bispecific molecules comprising atleast one first binding specificity for CD73 and a second bindingspecificity for a second target epitope. In an embodiment describedherein in which the bispecific molecule is multispecific, the moleculecan further include a third binding specificity.

In one embodiment, the bispecific molecules described herein comprise asa binding specificity at least one antibody, or an antibody fragmentthereof, including, e.g., an Fab, Fab′, F(ab′)₂, Fv, or a single chainFv. The antibody may also be a light chain or heavy chain dimer, or anyminimal fragment thereof such as a Fv or a single chain construct asdescribed in Ladner et al. U.S. Pat. No. 4,946,778, the contents ofwhich is expressly incorporated by reference.

Binding of the bispecific molecules to their specific targets can beconfirmed using art-recognized methods, such as enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis,bioassay (e.g., growth inhibition), or Western Blot assay. Each of theseassays generally detects the presence of protein-antibody complexes ofparticular interest by employing a labeled reagent (e.g., an antibody)specific for the complex of interest.

XVI. Compositions

Further provided are compositions, e.g., a pharmaceutical compositions,containing one or a combination of anti-CD73 antibodies, orantigen-binding portion(s) thereof, described herein, formulatedtogether with a pharmaceutically acceptable carrier. Such compositionsmay include one or a combination of (e.g., two or more different)antibodies, or immunoconjugates or bispecific molecules describedherein. For example, a pharmaceutical composition described herein cancomprise a combination of antibodies (or immunoconjugates orbispecifics) that bind to different epitopes on the target antigen orthat have complementary activities.

In certain embodiments, a composition comprises an anti-CD73 antibody ata concentration of at least 1 mg/ml, 5 mg/ml, 10 mg/ml, 50 mg/ml, 100mg/ml, 150 mg/ml, 200 mg/ml, 1-300 mg/ml, or 100-300 mg/ml.

Pharmaceutical compositions described herein also can be administered incombination therapy, i.e., combined with other agents. For example, thecombination therapy can include an anti-CD73 antibody described hereincombined with at least one other anti-cancer and/or T-cell stimulating(e.g., activating) agent. Examples of therapeutic agents that can beused in combination therapy are described in greater detail below in thesection on uses of the antibodies described herein.

In some embodiments, therapeutic compositions disclosed herein caninclude other compounds, drugs, and/or agents used for the treatment ofcancer. Such compounds, drugs, and/or agents can include, for example,chemotherapy drugs, small molecule drugs or antibodies that stimulatethe immune response to a given cancer. In some instances, therapeuticcompositions can include, for example, one or more of the agents listedin the section on combination therapies.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e., antibody,immunoconjugate, or bispecific molecule, may be coated in a material toprotect the compound from the action of acids and other naturalconditions that may inactivate the compound.

The pharmaceutical compounds described herein may include one or morepharmaceutically acceptable salts. A “pharmaceutically acceptable salt”refers to a salt that retains the desired biological activity of theparent compound and does not impart any undesired toxicological effects(see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19). Examplesof such salts include acid addition salts and base addition salts. Acidaddition salts include those derived from nontoxic inorganic acids, suchas hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic,phosphorous and the like, as well as from nontoxic organic acids such asaliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoicacids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromaticsulfonic acids and the like. Base addition salts include those derivedfrom alkaline earth metals, such as sodium, potassium, magnesium,calcium and the like, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition described herein also may include apharmaceutically acceptable anti-oxidant. Examples of pharmaceuticallyacceptable antioxidants include: (1) water soluble antioxidants, such asascorbic acid, cysteine hydrochloride, sodium bisulfate, sodiummetabisulfite, sodium sulfite and the like; (2) oil-solubleantioxidants, such as ascorbyl palmitate, butylated hydroxyanisole(BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate,alpha-tocopherol, and the like; and (3) metal chelating agents, such ascitric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaricacid, phosphoric acid, and the like.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions described herein includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositionsdescribed herein is contemplated. Supplementary active compounds canalso be incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated, and the particular mode of administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the composition which produces a therapeutic effect. Generally, outof one hundred percent, this amount will range from about 0.01 percentto about ninety-nine percent of active ingredient, preferably from about0.1 percent to about 70 percent, most preferably from about 1 percent toabout 30 percent of active ingredient in combination with apharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms described herein are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

For administration of the antibody, the dosage ranges from about 0.0001to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight.For example dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or withinthe range of 1-10 mg/kg. An exemplary treatment regime entailsadministration once per week, once every two weeks, once every threeweeks, once every four weeks, once a month, once every 3 months or onceevery three to 6 months.

In some methods, two or more monoclonal antibodies with differentbinding specificities are administered simultaneously, in which case thedosage of each antibody administered falls within the ranges indicated.Antibody is usually administered on multiple occasions. Intervalsbetween single dosages can be, for example, weekly, monthly, every threemonths or yearly. Intervals can also be irregular as indicated bymeasuring blood levels of antibody to the target antigen in the patient.In some methods, dosage is adjusted to achieve a plasma antibodyconcentration of about 1-1000 μg/ml and in some methods about 25-300μg/ml.

An antibody can be administered as a sustained release formulation, inwhich case less frequent administration is required. Dosage andfrequency vary depending on the half-life of the antibody in thepatient. In general, human antibodies show the longest half-life,followed by humanized antibodies, chimeric antibodies, and nonhumanantibodies. The dosage and frequency of administration can varydepending on whether the treatment is prophylactic or therapeutic. Inprophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated, and preferably until the patient shows partial orcomplete amelioration of symptoms of disease. Thereafter, the patientcan be administered a prophylactic regime.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions described herein may be varied so as to obtain an amount ofthe active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient. The selected dosagelevel will depend upon a variety of pharmacokinetic factors includingthe activity of the particular compositions described herein employed,or the ester, salt or amide thereof, the route of administration, thetime of administration, the rate of excretion of the particular compoundbeing employed, the duration of the treatment, other drugs, compoundsand/or materials used in combination with the particular compositionsemployed, the age, sex, weight, condition, general health and priormedical history of the patient being treated, and like factors wellknown in the medical arts.

A “therapeutically effective dosage” of an anti-CD73 antibody describedherein preferably results in a decrease in severity of disease symptoms,an increase in frequency and duration of disease symptom-free periods,or a prevention of impairment or disability due to the diseaseaffliction. In the context of cancer, a therapeutically effective dosepreferably prevents further deterioration of physical symptomsassociated with cancer. Symptoms of cancer are well-known in the art andinclude, for example, unusual mole features, a change in the appearanceof a mole, including asymmetry, border, color and/or diameter, a newlypigmented skin area, an abnormal mole, darkened area under nail, breastlumps, nipple changes, breast cysts, breast pain, death, weight loss,weakness, excessive fatigue, difficulty eating, loss of appetite,chronic cough, worsening breathlessness, coughing up blood, blood in theurine, blood in stool, nausea, vomiting, liver metastases, lungmetastases, bone metastases, abdominal fullness, bloating, fluid inperitoneal cavity, vaginal bleeding, constipation, abdominal distension,perforation of colon, acute peritonitis (infection, fever, pain), pain,vomiting blood, heavy sweating, fever, high blood pressure, anemia,diarrhea, jaundice, dizziness, chills, muscle spasms, colon metastases,lung metastases, bladder metastases, liver metastases, bone metastases,kidney metastases, and pancreatic metastases, difficulty swallowing, andthe like.

A therapeutically effective dose may prevent or delay onset of cancer,such as may be desired when early or preliminary signs of the diseaseare present. Laboratory tests utilized in the diagnosis of cancerinclude chemistries (including the measurement of CD73 levels),hematology, serology and radiology. Accordingly, any clinical orbiochemical assay that monitors any of the foregoing may be used todetermine whether a particular treatment is a therapeutically effectivedose for treating cancer. One of ordinary skill in the art would be ableto determine such amounts based on such factors as the subject's size,the severity of the subject's symptoms, and the particular compositionor route of administration selected.

A composition described herein can be administered via one or moreroutes of administration using one or more of a variety of methods knownin the art. As will be appreciated by the skilled artisan, the routeand/or mode of administration will vary depending upon the desiredresults. Preferred routes of administration for antibodies describedherein include intravenous, intramuscular, intradermal, intraperitoneal,subcutaneous, spinal or other parenteral routes of administration, forexample by injection or infusion. The phrase “parenteral administration”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

Alternatively, an antibody described herein can be administered via anon-parenteral route, such as a topical, epidermal or mucosal route ofadministration, for example, intranasally, orally, vaginally, rectally,sublingually or topically.

The active compounds can be prepared with carriers that will protect thecompound against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

Therapeutic compositions can be administered with medical devices knownin the art. For example, in a preferred embodiment, a therapeuticcomposition described herein can be administered with a needlelesshypodermic injection device, such as the devices disclosed in U.S. Pat.No. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or4,596,556. Examples of well-known implants and modules for use withanti-CD73 antibodies described herein include: U.S. Pat. No. 4,487,603,which discloses an implantable micro-infusion pump for dispensingmedication at a controlled rate; U.S. Pat. No. 4,486,194, whichdiscloses a therapeutic device for administering medicants through theskin; U.S. Pat. No. 4,447,233, which discloses a medication infusionpump for delivering medication at a precise infusion rate; U.S. Pat. No.4,447,224, which discloses a variable flow implantable infusionapparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, whichdiscloses an osmotic drug delivery system having multi-chambercompartments; and U.S. Pat. No. 4,475,196, which discloses an osmoticdrug delivery system. These patents are incorporated herein byreference. Many other such implants, delivery systems, and modules areknown to those skilled in the art.

In certain embodiments, the anti-CD73 antibodies described herein can beformulated to ensure proper distribution in vivo. For example, theblood-brain barrier (BBB) excludes many highly hydrophilic compounds. Toensure that the therapeutic compounds described herein cross the BBB (ifdesired), they can be formulated, for example, in liposomes. For methodsof manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811;5,374,548; and 5,399,331. The liposomes may comprise one or moremoieties which are selectively transported into specific cells ororgans, thus enhance targeted drug delivery (see, e.g., V. V. Ranade(1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties includefolate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.);mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun.153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140;M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactantprotein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134);p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K.Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I.J. Fidler (1994) Immunomethods 4:273.

XVII. Uses and Methods

The antibodies, antibody compositions and methods described herein havenumerous in vitro and in vivo applications, e.g., inhibiting tumorgrowth, inhibiting tumor metastasis, enhancing of immune response by,e.g., reducing adenosine signaling, or detection of CD73. In a preferredembodiment, the antibodies described herein are human antibodies. Forexample, anti-CD73 antibodies described herein can be administered tocells in culture, in vitro or ex vivo, or to human subjects, e.g., invivo, to inhibit tumor cell proliferation. Accordingly, provided hereinare methods of modifying tumor growth in a subject comprisingadministering to the subject an antibody, or antigen-binding portionthereof, described herein such that the tumor growth in the subject isreduced.

In a particular embodiment, the methods are particularly suitable fortreatment of cancer in vivo. To achieve antigen-specific inhibition oftumor growth, anti-CD73 antibodies described herein can be administeredtogether with an antigen of interest or the antigen may already bepresent in the subject to be treated (e.g., a tumor-bearing subject).When antibodies to CD73 are administered together with another agent,the two can be administered separately or simultaneously.

Also encompassed are methods for detecting the presence of human CD73antigen in a sample, or measuring the amount of human CD73 antigen,comprising contacting the sample, and a control sample, with a humanmonoclonal antibody, or an antigen binding portion thereof, whichspecifically binds to human CD73, under conditions that allow forformation of a complex between the antibody or portion thereof and humanCD73. The formation of a complex is then detected, wherein a differencecomplex formation between the sample compared to the control sample isindicative the presence of human CD73 antigen in the sample. Moreover,the anti-CD73 antibodies described herein can be used to purify humanCD73 via immunoaffinity purification.

Further encompassed are methods of stimulating an immune response (e.g.,an antigen-specific T cell response) in a subject comprisingadministering an anti-CD73 antibody described herein to the subject suchthat an immune response (e.g., an antigen-specific T cell response) inthe subject is stimulated. In a preferred embodiment, the subject is atumor-bearing subject and an immune response against the tumor isstimulated. A tumor may be a solid tumor or a liquid tumor, e.g., ahematological malignancy. In certain embodiments, a tumor is animmunogenic tumor. In certain embodiments, a tumor is non-immunogenic.

These and other methods described herein are discussed in further detailbelow.

Cancer

Inhibition of CD73 by anti-CD73 antibodies can reduce tumor growth andmetastasis in a patient. Inhibition of CD73 by anti-CD73 antibodies canalso enhance the immune response to cancerous cells in the patient.Provided herein are methods for treating a subject having cancer,comprising administering to the subject an anti-CD73 antibody describedherein, such that the subject is treated, e.g., such that growth ofcancerous tumors is inhibited or reduced and/or that the tumors regress.An anti-CD73 antibody can be used alone to inhibit the growth ofcancerous tumors. Alternatively, an anti-CD73 antibody can be used inconjunction with another agent, e.g., other immunogenic agents, standardcancer treatments, or other antibodies, as described below.

Accordingly, provided herein are methods of treating cancer, e.g., byinhibiting growth of tumor cells, in a subject, comprising administeringto the subject a therapeutically effective amount of an anti-CD73antibody described herein, or antigen-binding portion thereof. Theantibody may be a human anti-CD73 antibody (such as any of the humananti-human CD73 antibodies described herein). Additionally oralternatively, the antibody can be a chimeric or humanized anti-CD73antibody, e.g., a chimeric or humanized anti-CD73 antibody comprising ofan anti-CD73 antibody described herein, or antigen-binding portionthereof.

Cancers whose growth may be inhibited using the antibodies of theinvention include cancers typically responsive to immunotherapy.Non-limiting examples of cancers for treatment include squamous cellcarcinoma, small-cell lung cancer, non-small cell lung cancer, squamousnon-small cell lung cancer (NSCLC), non NSCLC, glioma, gastrointestinalcancer, renal cancer (e.g. clear cell carcinoma), ovarian cancer, livercancer, colorectal cancer, endometrial cancer, kidney cancer (e.g.,renal cell carcinoma (RCC)), prostate cancer (e.g. hormone refractoryprostate adenocarcinoma), thyroid cancer, neuroblastoma, pancreaticcancer, glioblastoma (glioblastoma multiforme), cervical cancer, stomachcancer, bladder cancer, hepatoma, breast cancer, colon carcinoma, andhead and neck cancer (or carcinoma), gastric cancer, germ cell tumor,pediatric sarcoma, sinonasal natural killer, melanoma (e.g., metastaticmalignant melanoma, such as cutaneous or intraocular malignantmelanoma), bone cancer, skin cancer, uterine cancer, cancer of the analregion, testicular cancer, carcinoma of the fallopian tubes, carcinomaof the endometrium, carcinoma of the cervix, carcinoma of the vagina,carcinoma of the vulva, cancer of the esophagus, cancer of the smallintestine, cancer of the endocrine system, cancer of the parathyroidgland, cancer of the adrenal gland, sarcoma of soft tissue, cancer ofthe urethra, cancer of the penis, solid tumors of childhood, cancer ofthe ureter, carcinoma of the renal pelvis, neoplasm of the centralnervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinalaxis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma,epidermoid cancer, squamous cell cancer, T-cell lymphoma,environmentally-induced cancers including those induced by asbestos,virus-related cancers (e.g., human papilloma virus (HPV)-related tumor),and hematologic malignancies derived from either of the two major bloodcell lineages, i.e., the myeloid cell line (which produces granulocytes,erythrocytes, thrombocytes, macrophages and mast cells) or lymphoid cellline (which produces B, T, NK and plasma cells), such as all types ofluekemias, lymphomas, and myelomas, e.g., acute, chronic, lymphocyticand/or myelogenous leukemias, such as acute leukemia (ALL), acutemyelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), andchronic myelogenous leukemia (CML), undifferentiated AML (MO),myeloblastic leukemia (M1), myeloblastic leukemia (M2; with cellmaturation), promyelocytic leukemia (M3 or M3 variant [M3V]),myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]),monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia(M7), isolated granulocytic sarcoma, and chloroma; lymphomas, such asHodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B-cell lymphomas,T-cell lymphomas, lymphoplasmacytoid lymphoma, monocytoid B-celllymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic(e.g., Ki 1+) large-cell lymphoma, adult T-cell lymphoma/leukemia,mantle cell lymphoma, angio immunoblastic T-cell lymphoma, angiocentriclymphoma, intestinal T-cell lymphoma, primary mediastinal B-celllymphoma, precursor T-lymphoblastic lymphoma, T-lymphoblastic; andlymphoma/leukaemia (T-Lbly/T-ALL), peripheral T-cell lymphoma,lymphoblastic lymphoma, post-transplantation lymphoproliferativedisorder, true histiocytic lymphoma, primary central nervous systemlymphoma, primary effusion lymphoma, lymphoblastic lymphoma (LBL),hematopoietic tumors of lymphoid lineage, acute lymphoblastic leukemia,diffuse large B-cell lymphoma, Burkitt's lymphoma, follicular lymphoma,diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma,precursor B-lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC)(also called mycosis fungoides or Sezary syndrome), andlymphoplasmacytoid lymphoma (LPL) with Waldenstrom's macroglobulinemia;myelomas, such as IgG myeloma, light chain myeloma, nonsecretorymyeloma, smoldering myeloma (also called indolent myeloma), solitaryplasmocytoma, and multiple myelomas, chronic lymphocytic leukemia (CLL),hairy cell lymphoma; hematopoietic tumors of myeloid lineage, tumors ofmesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma;seminoma, teratocarcinoma, tumors of the central and peripheral nervous,including astrocytoma, schwannomas; tumors of mesenchymal origin,including fibrosarcoma, rhabdomyoscaroma, and osteosarcoma; and othertumors, including melanoma, xeroderma pigmentosum, keratoacanthoma,seminoma, thyroid follicular cancer and teratocarcinoma, hematopoietictumors of lymphoid lineage, for example T-cell and B-cell tumors,including but not limited to T-cell disorders such as T-prolymphocyticleukemia (T-PLL), including of the small cell and cerebriform cell type;large granular lymphocyte leukemia (LGL) preferably of the T-cell type;a/d T-NHL hepatosplenic lymphoma; peripheral/post-thymic T cell lymphoma(pleomorphic and immunoblastic subtypes); angiocentric (nasal) T-celllymphoma; cancer of the head or neck, renal cancer, rectal cancer,cancer of the thyroid gland; acute myeloid lymphoma, as well as anycombinations of said cancers. The methods described herein may also beused for treatment of metastatic cancers, refractory cancers (e.g.,cancers refractory to previous immunotherapy, e.g., with a blockingCTLA-4 or PD-1 or PD-L1 antibody), and recurrent cancers.

The methods may be used for treating tumors or cancers that are CD73positive, or which express high levels of CD73. A method may comprisefirst determining the level of CD73 on tumors or tumor cells, andtreating with an anti-CD73 antibody, e.g, described herein, if thetumors or cells express CD73, e.g., high levels of CD73.

An anti-CD73 antibody can be administered as a monotherapy, or as theonly immunostimulating therapy. Antibodies to CD73, e.g., the anti-CD73antibodies described herein, can also be combined with an immunogenicagent, such as cancerous cells, purified tumor antigens (includingrecombinant proteins, peptides, and carbohydrate molecules), cells, andcells transfected with genes encoding immune stimulating cytokines (Heet al (2004) J. Immunol. 173:4919-28). Non-limiting examples of tumorvaccines that can be used include peptides of melanoma antigens, such aspeptides of gp100, MAGE antigens, Trp-2, MART1 and/or tyrosinase, ortumor cells transfected to express the cytokine GM-CSF (discussedfurther below).

In humans, some tumors have been shown to be immunogenic such asmelanomas. By lowering the threshold of T cell activation via CD73inhibition, the tumor responses in the host can be activated, allowingtreatment of non-immunogenic tumors or those having limitedimmunogenicity.

An anti-CD73 antibody, e.g., an anti-CD73 antibody described herein, maybe combined with a vaccination protocol. Many experimental strategiesfor vaccination against tumors have been devised (see Rosenberg, S.,2000, Development of Cancer Vaccines, ASCO Educational Book Spring:60-62; Logothetis, C., 2000, ASCO Educational Book Spring: 300-302;Khayat, D. 2000, ASCO Educational Book Spring: 414-428; Foon, K. 2000,ASCO Educational Book Spring: 730-738; see also Restifo, N. and Sznol,M., Cancer Vaccines, Ch. 61, pp. 3023-3043 in DeVita et al. (eds.),1997, Cancer: Principles and Practice of Oncology, Fifth Edition). Inone of these strategies, a vaccine is prepared using autologous orallogeneic tumor cells. These cellular vaccines have been shown to bemost effective when the tumor cells are transduced to express GM-CSF.GM-CSF has been shown to be a potent activator of antigen presentationfor tumor vaccination (Dranoff et al. (1993) Proc. Natl. Acad. SciU.S.A. 90: 3539-43).

The study of gene expression and large scale gene expression patterns invarious tumors has led to the definition of so called tumor specificantigens (Rosenberg, S A (1999) Immunity 10: 281-7). In many cases,these tumor specific antigens are differentiation antigens expressed inthe tumors and in the cell from which the tumor arose, for examplemelanocyte antigens gp100, MAGE antigens, and Trp-2. More importantly,many of these antigens can be shown to be the targets of tumor specificT cells found in the host. CD73 inhibition can be used in conjunctionwith a collection of recombinant proteins and/or peptides expressed in atumor in order to generate an immune response to these proteins. Theseproteins are normally viewed by the immune system as self antigens andare therefore tolerant to them. The tumor antigen can include theprotein telomerase, which is required for the synthesis of telomeres ofchromosomes and which is expressed in more than 85% of human cancers andin only a limited number of somatic tissues (Kim et al. (1994) Science266: 2011-2013). Tumor antigen can also be “neo-antigens” expressed incancer cells because of somatic mutations that alter protein sequence orcreate fusion proteins between two unrelated sequences (i.e., bcr-abl inthe Philadelphia chromosome), or idiotype from B cell tumors.

Other tumor vaccines can include the proteins from viruses implicated inhuman cancers such a Human Papilloma Viruses (HPV), Hepatitis Viruses(HBV and HCV) and Kaposi's Herpes Sarcoma Virus (KHSV). Another form oftumor specific antigen which can be used in conjunction with CD73inhibition is purified heat shock proteins (HSP) isolated from the tumortissue itself. These heat shock proteins contain fragments of proteinsfrom the tumor cells and these HSPs are highly efficient at delivery toantigen presenting cells for eliciting tumor immunity (Suot & Srivastava(1995) Science 269:1585-1588; Tamura et al. (1997) Science 278:117-120).

Dendritic cells (DC) are potent antigen presenting cells that can beused to prime antigen-specific responses. DC's can be produced ex vivoand loaded with various protein and peptide antigens as well as tumorcell extracts (Nestle et al. (1998) Nature Medicine 4: 328-332). DCs canalso be transduced by genetic means to express these tumor antigens aswell. DCs have also been fused directly to tumor cells for the purposesof immunization (Kugler et al. (2000) Nature Medicine 6:332-336). As amethod of vaccination, DC immunization can be effectively combined withCD73 inhibition to activate more potent anti-tumor responses.

CD73 inhibition can also be combined with standard cancer treatments(e.g., surgery, radiation, and chemotherapy). CD73 inhibition can beeffectively combined with chemotherapeutic regimes. In these instances,it may be possible to reduce the dose of chemotherapeutic reagentadministered (Mokyr et al. (1998) Cancer Research 58: 5301-5304). Anexample of such a combination is an anti-CD73 antibody in combinationwith decarbazine for the treatment of melanoma. Another example of sucha combination is an anti-CD73 antibody in combination with interleukin-2(IL-2) for the treatment of melanoma. The scientific rationale behindthe combined use of CD73 inhibition and chemotherapy is that cell death,that is a consequence of the cytotoxic action of most chemotherapeuticcompounds, should result in increased levels of tumor antigen in theantigen presentation pathway. Other combination therapies that mayresult in synergy with CD73 inhibition through cell death are radiation,surgery, and hormone deprivation. Each of these protocols creates asource of tumor antigen in the host. Angiogenesis inhibitors can also becombined with CD73 inhibition. Inhibition of angiogenesis leads to tumorcell death which may feed tumor antigen into host antigen presentationpathways.

Yet another example of such a combination is an anti-CD73 antibody incombination with an anti-CD39, anti-A2AR or chemical inhibitor, orantiA2BR antibody or chemical inhibitor. The scientific rational behindthe combined use of CD73 inhibition and inhibition of CD39, A2AR, orA2BR is that these proteins are also linked to CD73 biological functionand signaling. Specifically, CD39 catalyzes the conversion of ATP or ADPto AMP, thus providing the substrate (AMP) for CD73 enzymatic activity(i.e. the conversion of AMP to adenosine). Furthermore, adenosine is aligand for four known receptors, including AIR, A2AR, A2BR, and A3. A2ARand A2BR have been shown to regulate tumor cell proliferation, growth,migration, and metastasis, as well as T-cell activation in the tumorenvironment through cAMP signaling.

The anti-CD73 antibodies described herein can also be used incombination with bispecific antibodies that target Fcα or Fcγreceptor-expressing effectors cells to tumor cells (see, e.g., U.S. Pat.Nos. 5,922,845 and 5,837,243). Bispecific antibodies can be used totarget two separate antigens. For example anti-Fc receptor/anti tumorantigen (e.g., Her-2/neu) bispecific antibodies have been used to targetmacrophages to sites of tumor. This targeting may more effectivelyactivate tumor specific responses. Alternatively, antigen may bedelivered directly to DCs by the use of bispecific antibodies which bindto tumor antigen and a dendritic cell specific cell surface marker.

Tumors evade host immune surveillance by a large variety of mechanisms.Many of these mechanisms may be overcome by the inactivation of proteinswhich are expressed by the tumors and which are immunosuppressive. Theseinclude among others TGF-3 (Kehrl et al. (1986) J. Exp. Med. 163:1037-1050), IL-10 (Howard & O'Garra (1992) Immunology Today 13:198-200), and Fas ligand (Hahne et al. (1996) Science 274: 1363-1365).Antibodies to each of these entities can be used in combination withanti-CD73 antibodies to counteract the effects of the immunosuppressiveagent and favor tumor immune responses by the host.

Other antibodies which activate host immune responsiveness can be usedin combination with anti-CD73 antibodies. These include molecules on thesurface of dendritic cells which activate DC function and antigenpresentation. Anti-CD40 antibodies are able to substitute effectivelyfor T cell helper activity (Ridge et al. (1998) Nature 393: 474-478) andcan be used in conjunction with anti-CD73 antibodies. Activatingantibodies to T cell costimulatory molecules such as OX-40 (Weinberg etal. (2000) Immunol 164: 2160-2169), 4-1BB (Melero et al. (1997) NatureMedicine 3: 682-685 (1997), and ICOS (Hutloff et al. (1999) Nature 397:262-266) may also provide for increased levels of T cell activation.Inhibitors of PD1, PD-L1 or CTLA-4 (e.g., U.S. Pat. No. 5,811,097), mayalso be used in conjunction with an anti-CD73 antibody.

Other methods described herein are used to treat patients that have beenexposed to particular toxins or pathogens. Accordingly, another aspectdescribed herein provides a method of treating an infectious disease ina subject comprising administering to the subject an anti-CD73 antibody,or antigen-binding portion thereof, such that the subject is treated forthe infectious disease. Additionally or alternatively, the antibody canbe a chimeric or humanized antibody.

In all of the above methods, CD73 inhibition can be combined with otherforms of immunotherapy such as cytokine treatment (e.g., interferons,GM-CSF, G-CSF, IL-2), or bispecific antibody therapy, which provides forenhanced presentation of tumor antigens (see, e.g., Holliger (1993)Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak (1994) Structure2:1121-1123).

Combination Therapies

In addition to the combinations therapies provided above, anti-CD73antibodies described herein can also be used in combination therapy,e.g., for treating cancer, as described below.

Further provided herein are methods of combination therapy in which ananti-CD73 antibody is coadministered with one or more additional agents,e.g., antibodies, that are effective in stimulating immune responses tothereby further enhance, stimulate or upregulate immune responses in asubject.

Generally, an anti-CD73 antibody described herein can be combined with(i) an agonist of a co-stimulatory receptor and/or (ii) an antagonist ofan inhibitory signal on T cells, both of which result in amplifyingantigen-specific T cell responses (immune checkpoint regulators).

Most of the co-stimulatory and co-inhibitory molecules are members ofthe immunoglobulin super family (IgSF), and anti-CD73 antibodiesdescribed herein may be administered with an agent that targets a memberof the IgSF family to increase an immune response. One important familyof membrane-bound ligands that bind to co-stimulatory or co-inhibitoryreceptors is the B7 family, which includes B7-1, B7-2, B7-H1 (PD-L),B7-DC (PD-L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), and B7-H6.Another family of membrane bound ligands that bind to co-stimulatory orco-inhibitory receptors is the TNF family of molecules that bind tocognate TNF receptor family members, which include CD40 and CD40L,OX-40, OX-40L, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137, GITR,TRAIL/Apo2-L, TRAILR1/DR4, TRAILR2/DR5, TRAILR3, TRAILR4, OPG, RANK,RANKL, TWEAKR/Fn 14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LTβR,LIGHT, DcR3, HVEM, VEGI/TL1A, TRAMP/DR3, EDAR, EDA1, XEDAR, EDA2, TNFR1,Lymphotoxin α/TNFβ, TNFR2, TNFα, LTβR, Lymphotoxin α 1β2, FAS, FASL,RELT, DR6, TROY, NGFR (see, e.g., Tansey (2009) Drug Discovery Today00:1). T cell activation is also regulated by soluble cytokines. Thus,anti-CD73 antibodies can be used in combination with (i) antagonists (orinhibitors or blocking agents) of proteins of the IgSF family or B7family or the TNF family that inhibit T cell activation or antagonistsof cytokines that inhibit T cell activation (e.g., IL-6, IL-10, TGF-3,VEGF; “immunosuppressive cytokines”) and/or (ii) agonists of stimulatoryreceptors of the IgSF family, B7 family or the TNF family or ofcytokines that stimulate T cell activation, for stimulating an immuneresponse, e.g., for treating proliferative diseases, such as cancer.

For example, T cell responses can be stimulated by a combination ofanti-CD73 antibodies described herein, e.g., CD73.4-IgG2CS-IgG1.1f, andone or more of the following agents:

-   -   (1) An antagonist (inhibitor or blocking agent) of a protein        that inhibits T cell activation (e.g., immune checkpoint        inhibitors), such as CTLA-4, PD-1, PD-L1, PD-L2, and LAG-3, as        described above, and any of the following proteins: TIM-3,        Galectin 9, CEACAM-1, BTLA, CD69, Galectin-1, TIGIT, CD113,        GPR56, VISTA, 2B4, CD48, GARP, CD73, PD1H, LAIR, TIM-1, TIM-4,        CD39.    -   (2) An agonist of a protein that stimulates T cell activation,        such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, GITR, GITRL,        ICOS, ICOS-L, OX40, OX40L, CD70, CD27, CD40, DR3 and CD28H.

Exemplary agents that modulate one of the above proteins and may becombined with antagonist anti-CD73 antibodies, e.g., those describedherein, for treating cancer, include: Yervoy™ (ipilimumab) orTremelimumab (to CTLA-4), galiximab (to B7.1), BMS-936558 (to PD-1),CT-011 (to PD-1), MK-3475 (to PD-1), AMP224 (to B7DC), BMS-936559 (toB7-H1), MPDL3280A (to B7-H1), MEDI-570 (to ICOS), AMG557 (to B7H2),MGA271 (to B7H3), IMP321 (to LAG-3), BMS-663513 (to CD137), PF-05082566(to CD137), CDX-1127 (to CD27), anti-OX40 (Providence Health Services),huMAbOX40L (to OX40L), Atacicept (to TACI), CP-870893 (to CD40),Lucatumumab (to CD40), Dacetuzumab (to CD40), Muromonab-CD3 (to CD3),Ipilumumab (to CTLA-4).

Other molecules that can be combined with antagonist anti-CD73antibodies for the treatment of cancer include antagonists of inhibitoryreceptors on NK cells or agonists of activating receptors on NK cells.For example, anti-CD73 antagonist antibodies can be combined withantagonists of KIR (e.g., lirilumab).

T cell activation is also regulated by soluble cytokines, and anti-CD73antibodies may be administered to a subject, e.g., having cancer, withantagonists of cytokines that inhibit T cell activation or agonists ofcytokines that stimulate T cell activation.

In certain embodiments, anti-CD73 antibodies can be used in combinationwith (i) antagonists (or inhibitors or blocking agents) of proteins ofthe IgSF family or B7 family or the TNF family that inhibit T cellactivation or antagonists of cytokines that inhibit T cell activation(e.g., IL-6, IL-10, TGF-ß, VEGF; “immunosuppressive cytokines”) and/or(ii) agonists of stimulatory receptors of the IgSF family, B7 family orthe TNF family or of cytokines that stimulate T cell activation, forstimulating an immune response, e.g., for treating proliferativediseases, such as cancer.

Yet other agents for combination therapies include agents that inhibitor deplete macrophages or monocytes, including but not limited to CSF-1Rantagonists such as CSF-1R antagonist antibodies including RG7155(WO11/70024, WO11/107553, WO11/131407, WO13/87699, WO13/119716,WO13/132044) or FPA-008 (WO11/140249; WO13169264; WO14/036357).

Anti-CD73 antibodies may also be administered with agents that inhibitTGF-3 signaling.

Additional agents that may be combined with an anti-CD73 antibodyinclude agents that enhance tumor antigen presentation, e.g., dendriticcell vaccines, GM-CSF secreting cellular vaccines, CpG oligonucleotides,and imiquimod, or therapies that enhance the immunogenicity of tumorcells (e.g., anthracyclines).

Yet other therapies that may be combined with an anti-CD73 antibodyinclude therapies that deplete or block Treg cells, e.g., an agent thatspecifically binds to CD25.

Another therapy that may be combined with an anti-CD73 antibody is atherapy that inhibits a metabolic enzyme such as indoleamine dioxigenase(IDO), dioxigenase, arginase, or nitric oxide synthetase.

Another class of agents that may be used with an anti-CD73 antibodyincludes agents that inhibit the formation of adenosine or inhibit theadenosine A2A receptor.

Other therapies that may be combined with an anti-CD73 antibody fortreating cancer include therapies that reverse/prevent T cell anergy orexhaustion and therapies that trigger an innate immune activation and/orinflammation at a tumor site.

An anti-CD73 antibody may be combined with more than one immuno-oncologyagent, and may be, e.g., combined with a combinatorial approach thattargets multiple elements of the immune pathway, such as one or more ofthe following: a therapy that enhances tumor antigen presentation (e.g.,dendritic cell vaccine, GM-CSF secreting cellular vaccines, CpGoligonucleotides, imiquimod); a therapy that inhibits negative immuneregulation e.g., by inhibiting CTLA-4 and/or PD1/PD-L1/PD-L2 pathwayand/or depleting or blocking Tregs or other immune suppressing cells; atherapy that stimulates positive immune regulation, e.g., with agoniststhat stimulate the CD-137, OX-40, and/or GITR pathway and/or stimulate Tcell effector function; a therapy that increases systemically thefrequency of anti-tumor T cells; a therapy that depletes or inhibitsTregs, such as Tregs in the tumor, e.g., using an antagonist of CD25(e.g., daclizumab) or by ex vivo anti-CD25 bead depletion; a therapythat impacts the function of suppressor myeloid cells in the tumor; atherapy that enhances immunogenicity of tumor cells (e.g.,anthracyclines); adoptive T cell or NK cell transfer includinggenetically modified cells, e.g., cells modified by chimeric antigenreceptors (CAR-T therapy); a therapy that inhibits a metabolic enzymesuch as indoleamine dioxigenase (IDO), dioxigenase, arginase, or nitricoxide synthetase; a therapy that reverses/prevents T cell anergy orexhaustion; a therapy that triggers an innate immune activation and/orinflammation at a tumor site; administration of immune stimulatorycytokines; or blocking of immunorepressive cytokines.

Generally, anti-CD73 antibodies described herein can be used togetherwith one or more of agonistic agents that ligate positive costimulatoryreceptors, blocking agents that attenuate signaling through inhibitoryreceptors, antagonists, and one or more agents that increasesystemically the frequency of anti-tumor T cells, agents that overcomedistinct immune suppressive pathways within the tumor microenvironment(e.g., block inhibitory receptor engagement (e.g., PD-L1/PD-1interactions), deplete or inhibit Tregs (e.g., using an anti-CD25monoclonal antibody (e.g., daclizumab) or by ex vivo anti-CD25 beaddepletion), inhibit metabolic enzymes such as IDO, or reverse/prevent Tcell anergy or exhaustion) and agents that trigger innate immuneactivation and/or inflammation at tumor sites. An increasedinternalization of inhibitory receptors may translate into a lower levelof a potential inhibitor (assuming that signaling does not ensue).

In certain embodiments, an anti-CD73 antibody is administered to asubject together with a BRAF inhibitor if the subject is BRAF V600mutation positive.

Provided herein are methods for stimulating an immune response in asubject comprising administering to the subject an antagonist anti-CD73molecule, e.g., an antibody, and one or more additionalimmunostimulatory antibodies, such as an anti-PD-1 antagonist, e.g.,antagonist antibody, an anti-PD-L1 antagonist, e.g., antagonistantibody, an antagonist anti-CTLA-4 antagonist, e.g., antagonistantibody and/or an anti-LAG3 antagonist, e.g., an antagonist antibody,such that an immune response is stimulated in the subject, for exampleto inhibit tumor growth or to stimulate an anti-viral response. In oneembodiment, the subject is administered an antagonist anti-CD73 antibodyand an antagonist anti-PD-1 antibody. In one embodiment, the subject isadministered an antagonist anti-CD73 antibody and an antagonistanti-PD-L1 antibody. In one embodiment, the subject is administered anantagonist anti-CD73 antibody and an antagonist anti-CTLA-4 antibody. Inone embodiment, the anti-CD73 antibody is a human antibody, such as anantibody described herein. Alternatively, the anti-CD73 antibody can be,for example, a chimeric or humanized antibody (e.g., prepared from amouse anti-CD73 mAb), such as those further described herein. In oneembodiment, the at least one additional immunostimulatory antibody(e.g., an antagonist anti-PD-1, an antagonist anti-PD-L1, an antagonistanti-CTLA-4 and/or an antagonist anti-LAG3 antibody) is a humanantibody. Alternatively, the at least one additional immunostimulatoryantibody can be, for example, a chimeric or humanized antibody (e.g.,prepared from a mouse anti-PD-1, anti-PD-L1, anti-CTLA-4 and/oranti-LAG3 antibody).

Provided herein are methods for treating a hyperproliferative disease(e.g., cancer), comprising administering an antagonist anti-CD73antibody and an antagonist PD-1 antibody to a subject. In certainembodiments, the anti-CD73 antibody is administered at a subtherapeuticdose, the anti-PD-1 antibody is administered at a subtherapeutic dose,or both are administered at a subtherapeutic dose. Also provided hereinare methods for altering an adverse event associated with treatment of ahyperproliferative disease with an immunostimulatory agent, comprisingadministering an anti-CD73 antibody and a subtherapeutic dose ofanti-PD-1 antibody to a subject. In certain embodiments, the subject ishuman. In certain embodiments, the anti-PD-1 antibody is a humansequence monoclonal antibody and the anti-CD73 antibody is humansequence monoclonal antibody, such as an antibody comprising the CDRs orvariable regions of 11F11, 4C3, 4D4, 10D2, 11A6, 24H2, 5F8, 6E11, 7A11,CD73.3, CD73.4, CD73.5, CD73.6, CD73.7, CD73.8, CD73.9, CD73.10 orCD73.11 described herein or another antagonist anti-CD73 antibodydescribed herein.

Suitable PD-1 antagonists for use in the methods described herein,include, without limitation, ligands, antibodies (e.g., monoclonalantibodies and bispecific antibodies), and multivalent agents. In oneembodiment, the PD-1 antagonist is a fusion protein, e.g., an Fc fusionprotein, such as AMP-244. In one embodiment, the PD-1 antagonist is ananti-PD-1 or anti-PD-L1 antibody.

An exemplary anti-PD-1 antibody is nivolumab (BMS-936558) or an antibodythat comprises the CDRs or variable regions of one of antibodies 17D8,2D3, 4H1, 5C4, 7D3, 5F4 and 4A11 described in WO 2006/121168. In certainembodiments, an anti-PD1 antibody is MK-3475 (Lambrolizumab) describedin WO2012/145493; AMP-514 described in WO 2012/145493; and CT-011(Pidilizumab; previously CT-AcTibody or BAT; see, e.g., Rosenblatt etal. (2011) J. Immunotherapy 34:409). Further known PD-1 antibodies andother PD-1 inhibitors include those described in WO 2009/014708, WO03/099196, WO 2009/114335, WO 2011/066389, WO 2011/161699, WO2012/145493, U.S. Pat. Nos. 7,635,757 and 8,217,149, and U.S. PatentPublication No. 2009/0317368. Any of the anti-PD-1 antibodies disclosedin WO2013/173223 may also be used. An anti-PD-1 antibody that competesfor binding with, and/or binds to the same epitope on PD-1 as, as one ofthese antibodies may also be used in combination treatments.

In certain embodiments, the anti-PD-1 antibody binds to human PD-1 witha K_(D) of 5×10⁻⁸ M or less, binds to human PD-1 with a K_(D) of 1×10⁻⁸M or less, binds to human PD-1 with a K_(D) of 5×10⁻⁹ M or less, orbinds to human PD-1 with a K_(D) of between 1×10⁻⁸M and 1×10⁻¹⁰ M orless.

Provided herein are methods for treating a hyperproliferative disease(e.g., cancer), comprising administering an antagonist anti-CD73antibody and an antagonist PD-L1 antibody to a subject. In certainembodiments, the anti-CD73 antibody is administered at a subtherapeuticdose, the anti-PD-L1 antibody is administered at a subtherapeutic dose,or both are administered at a subtherapeutic dose. Provided herein aremethods for altering an adverse event associated with treatment of ahyperproliferative disease with an immunostimulatory agent, comprisingadministering an anti-CD73 antibody and a subtherapeutic dose ofanti-PD-L1 antibody to a subject. In certain embodiments, the subject ishuman. In certain embodiments, the anti-PD-L1 antibody is a humansequence monoclonal antibody and the anti-CD73 antibody is humansequence monoclonal antibody, such as an antibody comprising the CDRs orvariable regions of 11F11, 4C3, 4D4, 10D2, 11A6, 24H2, 5F8, 6E11, 7A11,CD73.3, CD73.4, CD73.5, CD73.6, CD73.7, CD73.8, CD73.9, CD73.10 orCD73.11 described herein or another antagonist anti-CD73 antibodydescribed herein.

In one embodiment, the anti-PD-L1 antibody is BMS-936559 (referred to as12A4 in WO 2007/005874 and U.S. Pat. No. 7,943,743), or an antibody thatcomprises the CDRs or variable regions of 3G10, 12A4, 10A5, 5F8, 10H10,1B12, 7H1, 11E6, 12B7 and 13G4, which are described in PCT PublicationWO 07/005874 and U.S. Pat. No. 7,943,743. In certain embodiment ananti-PD-L1 antibody is MEDI4736 (also known as Anti-B7-H1) or MPDL3280A(also known as RG7446). Any of the anti-PD-L1 antibodies disclosed inWO2013/173223, WO2011/066389, WO2012/145493, U.S. Pat. Nos. 7,635,757and 8,217,149 and U.S. Publication No. 2009/145493 may also be used.Anti-PD-L1 antibodies that compete with and/or bind to the same epitopeas that of any of these antibodies may also be used in combinationtreatments.

In certain embodiments, the anti-PD-L1 antibody binds to human PD-L1with a K_(D) of 5×10⁻⁸ M or less, binds to human PD-L1 with a K_(D) of1×10⁻⁸ M or less, binds to human PD-L1 with a K_(D) of 5×10⁻⁹ M or less,or binds to human PD-L1 with a K_(D) of between 1×10⁻⁸ M and 1×10⁻¹⁰ Mor less.

Provided herein are methods for treating a hyperproliferative disease(e.g., cancer), comprising administering an anti-CD73 antibody describedherein and a CTLA-4 antagonist antibody to a subject. In certainembodiments, the anti-CD73 antibody is administered at a subtherapeuticdose, the anti-CTLA-4 antibody is administered at a subtherapeutic dose,or both are administered at a subtherapeutic dose. Provided herein aremethods for altering an adverse event associated with treatment of ahyperproliferative disease with an immunostimulatory agent, comprisingadministering an anti-CD73 antibody and a subtherapeutic dose ofanti-CTLA-4 antibody to a subject. In certain embodiments, the subjectis human. In certain embodiments, the anti-CTLA-4 antibody is anantibody selected from the group of: Yervoy™ (ipilimumab or antibody10D1, described in PCT Publication WO 01/14424), tremelimumab (formerlyticilimumab, CP-675,206), monoclonal or an anti-CTLA-4 antibodydescribed in any of the following publications: WO 98/42752; WO00/37504; U.S. Pat. No. 6,207,156; Hurwitz et al. (1998) Proc. Natl.Acad. Sci. USA 95(17):10067-10071; Camacho et al. (2004) J. Clin.Oncology 22(145): Abstract No. 2505 (antibody CP-675206); and Mokyr etal. (1998) Cancer Res. 58:5301-5304. Any of the anti-CTLA-4 antibodiesdisclosed in WO2013/173223 may also be used.

In certain embodiments, the anti-CTLA-4 antibody binds to human CTLA-4with a K_(D) of 5×10⁻⁸ M or less, binds to human CTLA-4 with a K_(D) of1×10⁻⁸ M or less, binds to human CTLA-4 with a K_(D) of 5×10⁻⁹ M orless, or binds to human CTLA-4 with a K_(D) of between 1×10⁻⁸ M and1×10⁻¹⁰ M or less.

Provided herein are methods for treating a hyperproliferative disease(e.g., cancer), comprising administering an anti-CD73 antibody and ananti-LAG-3 antibody to a subject. In further embodiments, the anti-CD73antibody is administered at a subtherapeutic dose, the anti-LAG-3antibody is administered at a subtherapeutic dose, or both areadministered at a subtherapeutic dose. Provide herein are methods foraltering an adverse event associated with treatment of ahyperproliferative disease with an immunostimulatory agent, comprisingadministering an anti-CD73 antibody and a subtherapeutic dose ofanti-LAG-3 antibody to a subject. In certain embodiments, the subject ishuman. In certain embodiments, the anti-PD-L1 antibody is a humansequence monoclonal antibody and the anti-CD73 antibody is humansequence monoclonal antibody, such as an antibody comprising the CDRs orvariable regions of 11F11, 4C3, 4D4, 10D2, 11A6, 24H2, 5F8, 6E11, 7A11,CD73.3, CD73.4, CD73.5, CD73.6, CD73.7, CD73.8, CD73.9, CD73.10 orCD73.11 or another antagonist anti-CD73 antibody described herein.Examples of anti-LAG3 antibodies include antibodies comprising the CDRsor variable regions of antibodies 25F7, 26H10, 25E3, 8B7, 11F2 or 17E5,which are described in U.S. Patent Publication No. US2011/0150892 andWO2014/008218. In one embodiment, an anti-LAG-3 antibody is BMS-986016.Other art recognized anti-LAG-3 antibodies that can be used includeIMP731 described in US 2011/007023. IMP-321 may also be used. Anti-LAG-3antibodies that compete with and/or bind to the same epitope as that ofany of these antibodies may also be used in combination treatments.

In certain embodiments, the anti-LAG-3 antibody binds to human LAG-3with a K_(D) of 5×10⁻⁸ M or less, binds to human LAG-3 with a K_(D) of1×10⁻⁸ M or less, binds to human LAG-3 with a K_(D) of 5×10⁻⁹ M or less,or binds to human LAG-3 with a K_(D) of between 1×10⁻⁸ M and 1×10⁻¹⁰ Mor less.

In certain embodiments, the anti-CD73 antibody is administered togetherwith an anti-GITR agonist antibody, e.g., an antibody having the CDRsequences of 6C8, e.g., a humanized antibody having the CDRs of 6C8, asdescribed, e.g., in WO2006/105021; an antibody comprising the CDRs of ananti-GITR antibody described in WO2011/028683; an antibody comprisingthe CDRs of an anti-GITR antibody described in JP2008278814; or anantibody comprising the CDRs of an anti-GITR antibody described inPCT/US2015/033991.

Administration of anti-CD73 antibodies described herein and antagonists,e.g., antagonist antibodies, to one or more second target antigens suchas LAG-3 and/or CTLA-4 and/or PD-1 and/or PD-L1 can enhance the immuneresponse to cancerous cells in the patient. Cancers whose growth may beinhibited using the antibodies of the instant disclosure include cancerstypically responsive to immunotherapy. Representative examples ofcancers for treatment with the combination therapy of the instantdisclosure include those cancers specifically listed above in thediscussion of monotherapy with anti-CD73 antibodies.

In certain embodiments, the combination of therapeutic antibodiesdiscussed herein can be administered concurrently as a singlecomposition in a pharmaceutically acceptable carrier, or concurrently asseparate compositions with each antibody in a pharmaceuticallyacceptable carrier. In another embodiment, the combination oftherapeutic antibodies can be administered sequentially. For example, ananti-CTLA-4 antibody and an anti-CD73 antibody can be administeredsequentially, such as anti-CTLA-4 antibody being administered first andanti-CD73 antibody second, or anti-CD73 antibody being administeredfirst and anti-CTLA-4 antibody second. Additionally or alternatively, ananti-PD-1 antibody and an anti-CD73 antibody can be administeredsequentially, such as anti-PD-1 antibody being administered first andanti-CD73 antibody second, or anti-CD73 antibody being administeredfirst and anti-PD-1 antibody second. Additionally or alternatively, ananti-PD-L1 antibody and an anti-CD73 antibody can be administeredsequentially, such as anti-PD-L1 antibody being administered first andanti-CD73 antibody second, or anti-CD73 antibody being administeredfirst and anti-PD-L1 antibody second. Additionally or alternatively, ananti-LAG-3 antibody and an anti-CD73 antibody can be administeredsequentially, such as anti-LAG-3 antibody being administered first andanti-CD73 antibody second, or anti-CD73 antibody being administeredfirst and anti-LAG-3 antibody second.

Furthermore, if more than one dose of the combination therapy isadministered sequentially, the order of the sequential administrationcan be reversed or kept in the same order at each time point ofadministration, sequential administrations can be combined withconcurrent administrations, or any combination thereof. For example, thefirst administration of a combination anti-CTLA-4 antibody and anti-CD73antibody can be concurrent, the second administration can be sequentialwith anti-CTLA-4 antibody first and anti-CD73 antibody second, and thethird administration can be sequential with anti-CD73 antibody first andanti-CTLA-4 antibody second, etc. Additionally or alternatively, thefirst administration of a combination anti-PD-1 antibody and anti-CD73antibody can be concurrent, the second administration can be sequentialwith anti-PD-1 antibody first and anti-CD73 antibody second, and thethird administration can be sequential with anti-CD73 antibody first andanti-PD-1 antibody second, etc. Additionally or alternatively, the firstadministration of a combination anti-PD-L1 antibody and anti-CD73antibody can be concurrent, the second administration can be sequentialwith anti-PD-L1 antibody first and anti-CD73 antibody second, and thethird administration can be sequential with anti-CD73 antibody first andanti-PD-L1 antibody second, etc. Additionally or alternatively, thefirst administration of a combination anti-LAG-3 antibody and anti-CD73antibody can be concurrent, the second administration can be sequentialwith anti-LAG-3 antibody first and anti-CD73 antibody second, and thethird administration can be sequential with anti-CD73 antibody first andanti-LAG-3 antibody second, etc. Another representative dosing schemecan involve a first administration that is sequential with anti-CD73first and anti-CTLA-4 antibody (and/or anti-PD-1 antibody and/oranti-PD-L1 antibody and/or anti-LAG-3 antibody) second, and subsequentadministrations may be concurrent.

In one embodiment, a subject having a disease that may benefit fromstimulation of the immune system, e.g., cancer or an infectious disease,is treated by administration to the subject of an immune-oncology agentand an anti-CD73 antibody, wherein the immuno-oncology agent is a CD137(4-1BB) agonist, such as an agonistic CD137 antibody. Suitable CD137antibodies include, for example, urelumab or PF-05082566 (WO12/32433).

In one embodiment, a subject having a disease that may benefit fromstimulation of the immune system, e.g., cancer or an infectious disease,is treated by administration to the subject of an immune-oncology agentand an anti-CD73 antibody, wherein the immuno-oncology agent is an OX40agonist, such as an agonistic OX40 antibody. Suitable OX40 antibodiesinclude, for example, MEDI-6383, MEDI-6469 or MOXR0916 (RG7888;WO06/029879).

In one embodiment, a subject having a disease that may benefit fromstimulation of the immune system, e.g., cancer or an infectious disease,is treated by administration to the subject of an immune-oncology agentand an anti-CD73 antibody, wherein the immuno-oncology agent is a CD40agonist, such as an agonistic CD40 antibody. In certain embodiments, theimmuno-oncology agent is a CD40 antagonist, such as an antagonistic CD40antibody. Suitable CD40 antibodies include, for example, lucatumumab(HCD122), dacetuzumab (SGN-40), CP-870,893 or Chi Lob 7/4.

In one embodiment, a subject having a disease that may benefit fromstimulation of the immune system, e.g., cancer or an infectious disease,is treated by administration to the subject of an immune-oncology agentand an anti-CD73 antibody, wherein the immuno-oncology agent is a CD27agonist, such as an agonistic CD27 antibody. Suitable CD27 antibodiesinclude, for example, varlilumab (CDX-1127).

In one embodiment, a subject having a disease that may benefit fromstimulation of the immune system, e.g., cancer or an infectious disease,is treated by administration to the subject of an immune-oncology agentand an anti-CD73 antibody, wherein the immuno-oncology agent is MGA271(to B7H3) (WO11/109400).

In one embodiment, a subject having a disease that may benefit fromstimulation of the immune system, e.g., cancer or an infectious disease,is treated by administration to the subject of an immune-oncology agentand an anti-CD73 antibody, wherein the immuno-oncology agent is a KIRantagonist, such as lirilumab.

In one embodiment, a subject having a disease that may benefit fromstimulation of the immune system, e.g., cancer or an infectious disease,is treated by administration to the subject of an immune-oncology agentand an anti-CD73 antibody, wherein the immuno-oncology agent is an IDOantagonist. Suitable IDO antagonists include, for example, INCB-024360(WO2006/122150, WO07/75598, WO08/36653, WO08/36642), indoximod, NLG-919(WO09/73620, WO009/1156652, WO 11/56652, WO12/142237) or F001287.

In one embodiment, a subject having a disease that may benefit fromstimulation of the immune system, e.g., cancer or an infectious disease,is treated by administration to the subject of an immune-oncology agentand an anti-CD73 antibody, wherein the immuno-oncology agent is aToll-like receptor agonist, e.g., a TLR2/4 agonist (e.g., BacillusCalmette-Guerin); a TLR7 agonist (e.g., Hiltonol or Imiquimod); a TLR7/8agonist (e.g., Resiquimod); or a TLR9 agonist (e.g., CpG7909).

In one embodiment, a subject having a disease that may benefit fromstimulation of the immune system, e.g., cancer or an infectious disease,is treated by administration to the subject of an immune-oncology agentand an anti-CD73 antibody, wherein, the immuno-oncology agent is a TGF-3inhibitor, e.g., GC1008, LY2157299, TEW7197, or IMC-TR1.

In one aspect, an anti-CD73 antibody is sequentially administered priorto administration of a second agent, e.g., an immuno-oncology agent. Inone aspect, an anti-CD73 antibody is administered concurrently with thesecond agent, e.g., an immunology-oncology agent. In yet one aspect, ananti-CD73 antibody is sequentially administered after administration ofthe second agent. The administration of the two agents may start attimes that are, e.g., 30 minutes, 60 minutes, 90 minutes, 120 minutes, 3hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 5 days,7 days, or one or more weeks apart, or administration of the secondagent may start, e.g., 30 minutes, 60 minutes, 90 minutes, 120 minutes,3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 5days, 7 days, or one or more weeks after the first agent has beenadministered.

In certain aspects, an anti-CD73 antibody and a second agent, e.g., animmuno-oncology agent, are administered simultaneously, e.g., areinfused simultaneously, e.g., over a period of 30 or 60 minutes, to apatient. An anti-CD73 antibody may be co-formulated with a second agent,e.g., an immuno-oncology agent.

Optionally, an anti-CD73 as sole immunotherapeutic agent, or thecombination of an anti-CD73 antibody and one or more additionalimmunotherapeutic antibodies (e.g., anti-CTLA-4 and/or anti-PD-1 and/oranti-PD-L1 and/or anti-LAG-3 blockade) can be further combined with animmunogenic agent, such as cancerous cells, purified tumor antigens(including recombinant proteins, peptides, and carbohydrate molecules),cells, and cells transfected with genes encoding immune stimulatingcytokines (He et al. (2004) J. Immunol. 173:4919-28). Non-limitingexamples of tumor vaccines that can be used include peptides of melanomaantigens, such as peptides of gp100, MAGE antigens, Trp-2, MART1 and/ortyrosinase, or tumor cells transfected to express the cytokine GM-CSF(discussed further below). A combined CD73 inhibition and one or moreadditional antibodies (e.g., CTLA-4 and/or PD-1 and/or PD-L1 and/orLAG-3 blockade) can also be further combined with standard cancertreatments. For example, a combined CD73 inhibition and one or moreadditional antibodies (e.g., CTLA-4 and/or PD-1 and/or PD-L1 and/orLAG-3 blockade) can be effectively combined with chemotherapeuticregimes. In these instances, it is possible to reduce the dose of otherchemotherapeutic reagent administered with the combination of theinstant disclosure (Mokyr et al. (1998) Cancer Research 58: 5301-5304).An example of such a combination is a combination of anti-CD73antagonist antibody with or without and an additional antibody, such asanti-CTLA-4 antibodies and/or anti-PD-1 antibodies and/or anti-PD-L1antibodies and/or anti-LAG-3 antibodies) further in combination withdecarbazine for the treatment of melanoma. Another example is acombination of anti-CD73 antibody with or without and anti-CTLA-4antibodies and/or anti-PD-1 antibodies and/or anti-PD-L1 antibodiesand/or LAG-3 antibodies further in combination with interleukin-2 (IL-2)for the treatment of melanoma. The scientific rationale behind thecombined use of CD73 inhibition and CTLA-4 and/or PD-1 and/or PD-L1and/or LAG-3 blockade with chemotherapy is that cell death, which is aconsequence of the cytotoxic action of most chemotherapeutic compounds,should result in increased levels of tumor antigen in the antigenpresentation pathway. Other combination therapies that may result insynergy with a combined CD73 inhibition with or without and CTLA-4and/or PD-1 and/or PD-L1 and/or LAG-3 blockade through cell deathinclude radiation, surgery, or hormone deprivation. Each of theseprotocols creates a source of tumor antigen in the host. Angiogenesisinhibitors can also be combined with a combined CD73 inhibition andCTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3 blockade. Inhibition ofangiogenesis leads to tumor cell death, which can be a source of tumorantigen fed into host antigen presentation pathways.

An anti-CD73 antagonist antibody as sole immunotherapeutic agent, or acombination of CD73 antagonistic and CTLA-4 and/or PD-1 and/or PD-L1and/or LAG-3 blocking antibodies can also be used in combination withbispecific antibodies that target Fcα or Fcγ receptor-expressingeffector cells to tumor cells (see, e.g., U.S. Pat. Nos. 5,922,845 and5,837,243).

Bispecific antibodies can be used to target two separate antigens. The Tcell arm of these responses would be augmented by the use of a combinedCD73 inhibition and CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3blockade.

In another example, an anti-CD73 antagonist antibody as soleimmunotherapeutic agent or a combination of an anti-CD73 antibody andadditional immunostimulating agent, e.g., anti-CTLA-4 antibody and/oranti-PD-1 antibody and/or anti-PD-L1 antibody and/or LAG-3 agent, e.g.,antibody, can be used in conjunction with an anti-neoplastic antibody,such as Rituxan® (rituximab), Herceptin® (trastuzumab), Bexxar®(tositumomab), Zevalin® (ibritumomab), Campath® (alemtuzumab),Lymphocide® (eprtuzumab), Avastin® (bevacizumab), and Tarceva®(erlotinib), and the like. By way of example and not wishing to be boundby theory, treatment with an anti-cancer antibody or an anti-cancerantibody conjugated to a toxin can lead to cancer cell death (e.g.,tumor cells) which would potentiate an immune response mediated by theimmunostimulating agent, e.g., CD73, CTLA-4, PD-1, PD-L1 or LAG-3 agent,e.g., antibody. In an exemplary embodiment, a treatment of ahyperproliferative disease (e.g., a cancer tumor) can include ananti-cancer agent, e.g., antibody, in combination with anti-CD73 andoptionally an additional immunostimulating agent, e.g., anti-CTLA-4and/or anti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3 agent, e.g.,antibody, concurrently or sequentially or any combination thereof, whichcan potentiate an anti-tumor immune responses by the host.

Tumors evade host immune surveillance by a large variety of mechanisms.Many of these mechanisms may be overcome by the inactivation ofproteins, which are expressed by the tumors and which areimmunosuppressive. These include, among others, TGF-3 (Kehrl et al.(1986) J. Exp. Med. 163: 1037-1050), IL-10 (Howard & O'Garra (1992)Immunology Today 13: 198-200), and Fas ligand (Hahne et al. (1996)Science 274: 1363-1365). Antibodies to each of these entities can befurther combined with an anti-CD73 antibody with or without anadditional immunostimulating agent, e.g., an anti-CTLA-4 and/oranti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3 agent, such as antibody,to counteract the effects of immunosuppressive agents and favoranti-tumor immune responses by the host.

Other agents, e.g., antibodies, that can be used to activate host immuneresponsiveness can be further used in combination with an anti-CD73antibody with or without an additional immunostimulating agent, such asanti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3antibody. These include molecules on the surface of dendritic cells thatactivate DC function and antigen presentation. Anti-CD40 antibodies(Ridge et al., supra) can be used in conjunction with an anti-CD73antibody and optionally an additional immunostimulating agent, e.g., ananti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3 agent,e.g., antibody. Other activating antibodies to T cell costimulatorymolecules Weinberg et al., supra, Melero et al. supra, Hutloff et al.,supra, may also provide for increased levels of T cell activation.

As discussed above, bone marrow transplantation is currently being usedto treat a variety of tumors of hematopoietic origin. Anti-CD73immunotherapy alone or combined with CTLA-4 and/or PD-1 and/or PD-L1and/or LAG-3 blockade can be used to increase the effectiveness of thedonor engrafted tumor specific T cells.

Several experimental treatment protocols involve ex vivo activation andexpansion of antigen specific T cells and adoptive transfer of thesecells into recipients in order to antigen-specific T cells against tumor(Greenberg & Riddell, supra). These methods can also be used to activateT cell responses to infectious agents such as CMV. Ex vivo activation inthe presence of anti-CD73 with or without an additionalimmunostimulating therapy, e.g., anti-CTLA-4 and/or anti-PD-1 and/oranti-PD-L1 and/or anti-LAG-3 antibodies can be expected to increase thefrequency and activity of the adoptively transferred T cells.

Provided herein are methods for altering an adverse event associatedwith treatment of a hyperproliferative disease (e.g., cancer) with animmunostimulatory agent, comprising administering an anti-CD73 antibodywith or without and a subtherapeutic dose of anti-CTLA-4 and/oranti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3 agent, e.g., antibody, toa subject. For example, the methods described herein provide for amethod of reducing the incidence of immunostimulatory therapeuticantibody-induced colitis or diarrhea by administering a non-absorbablesteroid to the patient. As used herein, a “non-absorbable steroid” is aglucocorticoid that exhibits extensive first pass metabolism such that,following metabolism in the liver, the bioavailability of the steroid islow, i.e., less than about 20%. In one embodiment described herein, thenon-absorbable steroid is budesonide. Budesonide is a locally-actingglucocorticosteroid, which is extensively metabolized, primarily by theliver, following oral administration. ENTOCORT EC® (Astra-Zeneca) is apH- and time-dependent oral formulation of budesonide developed tooptimize drug delivery to the ileum and throughout the colon. ENTOCORTEC® is approved in the U.S. for the treatment of mild to moderateCrohn's disease involving the ileum and/or ascending colon. The usualoral dosage of ENTOCORT EC® for the treatment of Crohn's disease is 6 to9 mg/day. ENTOCORT EC® is released in the intestines before beingabsorbed and retained in the gut mucosa. Once it passes through the gutmucosa target tissue, ENTOCORT EC® is extensively metabolized by thecytochrome P450 system in the liver to metabolites with negligibleglucocorticoid activity. Therefore, the bioavailability is low (about10%). The low bioavailability of budesonide results in an improvedtherapeutic ratio compared to other glucocorticoids with less extensivefirst-pass metabolism. Budesonide results in fewer adverse effects,including less hypothalamic-pituitary suppression, thansystemically-acting corticosteroids. However, chronic administration ofENTOCORT EC® can result in systemic glucocorticoid effects such ashypercorticism and adrenal suppression. See PDR 58^(th)ed. 2004;608-610.

In still further embodiments, a CD73 inhibition with or without CTLA-4and/or PD-1 and/or PD-L1 and/or LAG-3 blockade (i.e., immunostimulatorytherapeutic antibodies anti-CD73 and optionally anti-CTLA-4 and/oranti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3 antibodies) in conjunctionwith a non-absorbable steroid can be further combined with a salicylate.Salicylates include 5-ASA agents such as, for example: sulfasalazine(AZULFIDINE®, Pharmacia & UpJohn); olsalazine (DIPENTUM®, Pharmacia &UpJohn); balsalazide (COLAZAL®, Salix Pharmaceuticals, Inc.); andmesalamine (ASACOL®, Procter & Gamble Pharmaceuticals; PENTASA®, ShireUS; CANASA®, Axcan Scandipharm, Inc.; ROWASA®, Solvay).

In accordance with the methods described herein, a salicylateadministered in combination with anti-CD73 with or without anti-CTLA-4and/or anti-PD-1 and/or anti-PD-L1 and/or LAG-3 antibodies and anon-absorbable steroid can includes any overlapping or sequentialadministration of the salicylate and the non-absorbable steroid for thepurpose of decreasing the incidence of colitis induced by theimmunostimulatory antibodies. Thus, for example, methods for reducingthe incidence of colitis induced by the immunostimulatory antibodiesdescribed herein encompass administering a salicylate and anon-absorbable concurrently or sequentially (e.g., a salicylate isadministered 6 hours after a non-absorbable steroid), or any combinationthereof. Further, a salicylate and a non-absorbable steroid can beadministered by the same route (e.g., both are administered orally) orby different routes (e.g., a salicylate is administered orally and anon-absorbable steroid is administered rectally), which may differ fromthe route(s) used to administer the anti-CD73 and anti-CTLA-4 and/oranti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3 antibodies.

The anti-CD73 antibodies and combination antibody therapies describedherein may also be used in conjunction with other well known therapiesthat are selected for their particular usefulness against the indicationbeing treated (e.g., cancer). Combinations of the anti-CD73 antibodiesdescribed herein may be used sequentially with known pharmaceuticallyacceptable agent(s).

For example, the anti-CD73 antibodies and combination antibody therapiesdescribed herein can be used in combination (e.g., simultaneously orseparately) with an additional treatment, such as irradiation,chemotherapy (e.g., using camptothecin (CPT-11), 5-fluorouracil (5-FU),cisplatin, doxorubicin, irinotecan, paclitaxel, gemcitabine, cisplatin,paclitaxel, carboplatin-paclitaxel (Taxol), doxorubicin, 5-fu, orcamptothecin+apo21/TRAIL (a 6× combo)), one or more proteasomeinhibitors (e.g., bortezomib or MG132), one or more Bcl-2 inhibitors(e.g., BH3I-2′ (bcl-xl inhibitor), indoleamine dioxygenase-1 (IDO1)inhibitor (e.g., INCB24360), AT-101 (R-(−)-gossypol derivative), ABT-263(small molecule), GX-15-070 (obatoclax), or MCL-1 (myeloid leukemia celldifferentiation protein-1) antagonists), iAP (inhibitor of apoptosisprotein) antagonists (e.g., smac7, smac4, small molecule smac mimetic,synthetic smac peptides (see Fulda et al., Nat Med 2002; 8:808-15),ISIS23722 (LY2181308), or AEG-35156 (GEM-640)), HDAC (histonedeacetylase) inhibitors, anti-CD20 antibodies (e.g., rituximab),angiogenesis inhibitors (e.g., bevacizumab), anti-angiogenic agentstargeting VEGF and VEGFR (e.g., Avastin), synthetic triterpenoids (seeHyer et al., Cancer Research 2005; 65:4799-808), c-FLIP (cellularFLICE-inhibitory protein) modulators (e.g., natural and syntheticligands of PPARy (peroxisome proliferator-activated receptor γ), 5809354or 5569100), kinase inhibitors (e.g., Sorafenib), Trastuzumab,Cetuximab, Temsirolimus, mTOR inhibitors such as rapamycin andtemsirolimus, Bortezomib, JAK2 inhibitors, HSP90 inhibitors, PI3K-AKTinhibitors, Lenalildomide, GSK3β inhibitors, IAP inhibitors and/orgenotoxic drugs.

The anti-CD73 antibodies and combination antibody therapies describedherein can further be used in combination with one or moreanti-proliferative cytotoxic agents. Classes of compounds that may beused as anti-proliferative cytotoxic agents include, but are not limitedto, the following: Alkylating agents (including, without limitation,nitrogen mustards, ethylenimine derivatives, alkyl sulfonates,nitrosoureas and triazenes): Uracil mustard, Chlormethine,Cyclophosphamide (CYTOXAN™) fosfamide, Melphalan, Chlorambucil,Pipobroman, Triethylenemelamine, Triethylenethiophosphoramine, Busulfan,Carmustine, Lomustine, Streptozocin, Dacarbazine, and Temozolomide.

Antimetabolites (including, without limitation, folic acid antagonists,pyrimidine analogs, purine analogs and adenosine deaminase inhibitors):Methotrexate, 5-Fluorouracil, Floxuridine, Cytarabine, 6-Mercaptopurine,6-Thioguanine, Fludarabine phosphate, Pentostatine, and Gemcitabine.

Suitable anti-proliferative agents for combining with antagonistanti-CD73 antibodies, without limitation, taxanes, paclitaxel(paclitaxel is commercially available as TAXOL™), docetaxel,discodermolide (DDM), dictyostatin (DCT), Peloruside A, epothilones,epothilone A, epothilone B, epothilone C, epothilone D, epothilone E,epothilone F, furanoepothilone D, desoxyepothilone Bl,[17]-dehydrodesoxyepothilone B, [18]dehydrodesoxyepothilones B,C12,13-cyclopropyl-epothilone A, C6-C8 bridged epothilone A,trans-9,10-dehydroepothilone D, cis-9,10-dehydroepothilone D,16-desmethylepothilone B, epothilone B10, discoderomolide, patupilone(EPO-906), KOS-862, KOS-1584, ZK-EPO, ABJ-789, XAA296A (Discodermolide),TZT-1027 (soblidotin), ILX-651 (tasidotin hydrochloride), HalichondrinB, Eribulin mesylate (E-7389), Hemiasterlin (HTI-286), E-7974,Cyrptophycins, LY-355703, Maytansinoid immunoconjugates (DM-1), MKC-1,ABT-751, T1-38067, T-900607, SB-715992 (ispinesib), SB-743921, MK-0731,STA-5312, eleutherobin,17beta-acetoxy-2-ethoxy-6-oxo-B-homo-estra-1,3,5(10)-trien-3-ol,cyclostreptin, isolaulimalide, laulimalide,4-epi-7-dehydroxy-14,16-didemethyl-(+)-discodermolides, andcryptothilone 1, in addition to other microtubuline stabilizing agentsknown in the art.

In cases where it is desirable to render aberrantly proliferative cellsquiescent in conjunction with or prior to treatment with anti-CD73antibodies described herein, hormones and steroids (including syntheticanalogs), such as 17a-Ethinylestradiol, Diethylstilbestrol,Testosterone, Prednisone, Fluoxymesterone, Dromostanolone propionate,Testolactone, Megestrolacetate, Methylprednisolone, Methyl-testosterone,Prednisolone, Triamcinolone, Chlorotrianisene, Hydroxyprogesterone,Aminoglutethimide, Estramustine, Medroxyprogesteroneacetate, Leuprolide,Flutamide, Toremifene, ZOLADEX™, can also be administered to thepatient. When employing the methods or compositions described herein,other agents used in the modulation of tumor growth or metastasis in aclinical setting, such as antimimetics, can also be administered asdesired.

Methods for the safe and effective administration of chemotherapeuticagents are known to those skilled in the art. In addition, theiradministration is described in the standard literature. For example, theadministration of many of the chemotherapeutic agents is described inthe Physicians' Desk Reference (PDR), e.g., 1996 edition (MedicalEconomics Company, Montvale, N.J. 07645-1742, USA); the disclosure ofwhich is incorporated herein by reference thereto.

The chemotherapeutic agent(s) and/or radiation therapy can beadministered according to therapeutic protocols well known in the art.It will be apparent to those skilled in the art that the administrationof the chemotherapeutic agent(s) and/or radiation therapy can be varieddepending on the disease being treated and the known effects of thechemotherapeutic agent(s) and/or radiation therapy on that disease.Also, in accordance with the knowledge of the skilled clinician, thetherapeutic protocols (e.g., dosage amounts and times of administration)can be varied in view of the observed effects of the administeredtherapeutic agents on the patient, and in view of the observed responsesof the disease to the administered therapeutic agents.

EXEMPLARY EMBODIMENTS

1. An isolated human antibody, or antigen binding portion thereof, whichbinds to human Cluster of Differentiation 73 (CD73) and exhibits one ormore of the following properties:

(a) inhibits CD73 enzymatic activity;

(b) internalizes into tumor cells or

(c) binds to a conformation epitope comprising amino acids 65-83 and157-172 of human CD73.

2. The antibody, or antigen binding portion thereof, of embodiment 1,wherein the antibody internalizes into tumor cells with a T_(1/2) of nomore than 10 min as measured by Pulse Chase.3. The antibody, or antigen binding portion thereof, of embodiment 1 or2, wherein the antibody binds to soluble human CD73 with a K_(D) ofabout 0.1 to 10 nM or less as measured by BIACORE® SPR analysis.4. The antibody, or antigen binding portion thereof, of any one of thepreceding embodiments, wherein the antibody binds to human CD73 with anEC₅₀ of 0.1 to 10 nM or less as measured by FACS.5. The antibody, or antigen binding portion thereof, of any one of thepreceding embodiments, wherein the antibody binds to cynomolgus CD73with an EC₅₀ of 0.1 to 10 nM or less as measured by FACS.6. The antibody, or antigen binding portion thereof, of any one of thepreceding embodiments, wherein the antibody binds to an epitope on humanCD73 (SEQ ID NO: 1) which includes amino acid residuesFTKVQQIRRAEPNVLLLDA (SEQ ID NO: 96) and/or LYLPYKVLPVGDEVVG (SEQ ID NO:97).7. The antibody, or antigen binding portion thereof, of embodiment 6,wherein the epitope spans or overlaps with amino acid residuesFTKVQQIRRAEPNVLLLDA (SEQ ID NO: 96) and/or LYLPYKVLPVGDEVVG (SEQ ID NO:97).8. The antibody, or antigen binding portion thereof, of any one of thepreceding embodiments, wherein the antibody is selected from the groupconsisting of an IgG1, an IgG2, an IgG3, an IgG4 or a variant thereof.9. An isolated monoclonal antibody, or antigen binding portion thereof,which binds to human CD73 and comprises three heavy chain variableregion CDRs and three light chain variable region CDRs that arerespectively in the heavy and light chain variable region pairs selectedfrom the group consisting of:

(a) SEQ ID NOs: 4 and 8

(b) SEQ ID NOs: 4 and 12;

(c) SEQ ID NOs: 16 and 20;

(d) SEQ ID NOs: 16 and 24;

(e) SEQ ID NOs: 16 and 28;

(f) SEQ ID NOs: 32 and 36;

(g) SEQ ID NOs: 40 and 44;

(h) SEQ ID NOs: 40 and 48;

(i) SEQ ID NOs: 52 and 56;

(j) SEQ ID NOs: 60 and 64;

(k) SEQ ID NOs: 68 and 72;

(l) SEQ ID NOs: 68 and 76;

(m) SEQ ID NOs: 80 and 84;

(n) SEQ ID NOs: 88 and 92;

(o) SEQ ID NOs: 135 and 8; and

(p) SEQ ID NOs: 135 and 12.

10. An isolated monoclonal antibody, or antigen binding portion thereof,which binds to human CD73, comprising:

(a) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 5,6, and 7, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 9, 10, and 11, respectively;

(b) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 5,6, and 7, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 13, 14, and 15, respectively;

(c) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:17, 18, and 19, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 21, 22, and 23, respectively;

(d) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:17, 18, and 19, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 25, 26, and 27, respectively;

(e) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:17, 18, and 19, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 29, 30, and 31, respectively;

(f) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:33, 34, and 35, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 37, 38, and 39, respectively;

(g) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:41, 42, and 43, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 45, 46, and 47, respectively;

(h) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:41, 42, and 43, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 49, 50, and 51, respectively;

(i) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:53, 54, and 55, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 57, 58, and 59, respectively;

(j) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:61, 62, and 63, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 65, 66, and 67, respectively;

(k) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:69, 70, and 71, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 73, 74, and 75, respectively;

(l) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:69, 70, and 71, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 77, 78, and 79, respectively;

(m) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:81, 82, and 83, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 85, 86, and 87, respectively; or

(n) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:89, 90, and 91, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 93, 94, and 95, respectively.

11. The antibody, or antigen binding portion thereof, of embodiment 10,wherein the antibody comprises heavy chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 5, 6, and 7, respectively, and/or lightchain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 9, 10, and11, respectively.12. The antibody, or antigen binding portion thereof, of embodiment 11,wherein the antibody comprises heavy chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 5, 6, and 7, respectively, and/or lightchain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 13, 14, and15, respectively.13. An isolated monoclonal antibody, or antigen binding portion thereof,which binds to human CD73 and comprises heavy and light chain variableregions, wherein the heavy chain variable region comprises an amino acidsequence which is at least 90% identical to the amino acid sequenceselected from the group consisting of SEQ ID NOs: 4, 16, 32, 40, 52, 60,68, 80, 88, and 135.14. An isolated monoclonal antibody, or antigen binding portion thereof,which binds to human CD73 and comprises heavy and light chain variableregions, wherein the light chain variable region comprises an amino acidsequence which is at least 90% identical to the amino acid sequenceselected from the group consisting of SEQ ID NOs: 8, 12, 20, 24, 28, 36,44, 48, 56, 64, 72, 76, 84, and 92.15. An isolated monoclonal antibody, or antigen binding portion thereof,which binds to human CD73 and comprises heavy and light chain variableregions which are at least 85% identical to the heavy and light chainvariable region amino acid sequences, respectively selected from thegroup consisting of:

(a) SEQ ID NOs: 4 and 8

(b) SEQ ID NOs: 4 and 12;

(c) SEQ ID NOs: 16 and 20;

(d) SEQ ID NOs: 16 and 24;

(e) SEQ ID NOs: 16 and 28;

(f) SEQ ID NOs: 32 and 36;

(g) SEQ ID NOs: 40 and 44;

(h) SEQ ID NOs: 40 and 48;

(i) SEQ ID NOs: 52 and 56;

(j) SEQ ID NOs: 60 and 64;

(k) SEQ ID NOs: 68 and 72;

(l) SEQ ID NOs: 68 and 76;

(m) SEQ ID NOs: 80 and 84;

(n) SEQ ID NOs: 88 and 92;

(o) SEQ ID NOs: 135 and 8; and

(p) SEQ ID NOs: 135 and 12.

16. The antibody, or antigen binding portion thereof, of embodiment 15,wherein the heavy and light chain variable regions comprise an aminoacid sequence at least 90% identical to the heavy and light chainvariable regions, respectively, selected from the group consisting of(a)-(p).17. The antibody, or antigen binding portion thereof, of embodiment 16,wherein the heavy and light chain variable regions comprise an aminoacid sequence at least 95% identical to the heavy and light chainvariable regions, respectively, selected from the group consisting of(a)-(p).18. The antibody, or antigen binding portion thereof, of embodiment 17,wherein the heavy and light chain variable regions comprise the heavyand light chain variable regions, respectively, selected from the groupconsisting of (a)-(p).19. The antibody, or antigen binding portion thereof, of embodiment 18,wherein the antibody comprises a heavy chain variable region comprisingthe amino acid sequence set forth in SEQ ID NO: 135 and a light chainvariable region comprising the amino acid sequence set forth in SEQ IDNO: 8.20. The antibody, or antigen binding portion thereof, of embodiment 18,wherein the antibody comprises a heavy chain variable region comprisingthe amino acid sequence set forth in SEQ ID NO: 135 and a light chainvariable region comprising the amino acid sequence set forth in SEQ IDNO: 12.21. An isolated monoclonal antibody, or antigen binding portion thereof,which binds to the same epitope on CD73 as the antibody of any one ofembodiments 1-20.22. The antibody, or antigen binding portion thereof, of any one ofembodiments 9-21, wherein the antibody exhibits any one of the followingproperties:

(1) binding to soluble human CD73, e.g., with a K_(D) of 10 nM or less(e.g., 0.01 nM to 10 nM), e.g., as measured by BIACORE® SPR analysis;

(2) binding to membrane bound human CD73, e.g., with an EC₅₀ of 1 nM orless (e.g., 0.01 nM to 1 nM);

(3) binding to cynomolgus CD73, e.g., bind to membrane bound cynomolgusCD73, e.g, with an EC₅₀ of 10 nM or less (e.g., 0.01 nM to 10 nM);

(4) inhibition of human CD73 enzymatic activity, e.g., with an EC50 of10 nM or less;

(5) inhibition of cyno CD73 enzymatic activity, e.g., with an EC50 of 10nM or less;

(6) inhibition of human CD73 enzymatic activity in vivo; inducing orenhancing T cell activation without requiring multivalent cross-linking;

(7); internalization into cells, e.g., with a T_(1/2) of less than 10minutes;

(8) binding to a conformational epitope on human CD73, e.g., adiscontinuous epitope within the amino acid sequence (SEQ ID NO: 1)which includes amino acid residues FTKVQQIRRAEPNVLLLDA (SEQ ID NO: 96)and/or LYLPYKVLPVGDEVVG (SEQ ID NO: 97)

(9) binding to glycosylated but not unglycosylated human CD73; and

(10) competing in either direction or both directions for binding tohuman CD73 with CD73.4-1, CD73.4-2, CD73.3, 11F11-1, 11F11-2, 4C3-1,4C3-2, 4C3-3, 4D4, 10D2-1, 10D2-2, 11A6, 24H2, 5F8-1, 5F8-2, 6E11 and/or7A11.

23. An isolated monoclonal antibody, or antigen binding portion thereof,which binds to CD73 and comprises heavy chain and light chain sequenceswhich are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical tothe amino acid sequences of the heavy and light chain sequences,respectively, selected from the group consisting of:

(a) SEQ ID NOs: 100 and 101, respectively;

(b) SEQ ID NOs: 100 and 102, respectively;

(c) SEQ ID NOs: 103 and 104, respectively;

(d) SEQ ID NOs: 103 and 105, respectively;

(e) SEQ ID NOs: 103 and 106, respectively;

(f) SEQ ID NOs: 107 and 108, respectively;

(g) SEQ ID NOs: 109 and 110, respectively;

(h) SEQ ID NOs: 109 and 111, respectively;

(i) SEQ ID NOs: 112 and 113, respectively;

(j) SEQ ID NOs: 114 and 115, respectively;

(k) SEQ ID NOs: 116 and 117, respectively;

(l) SEQ ID NOs: 116 and 118, respectively;

(m) SEQ ID NOs: 119 and 120, respectively;

(n) SEQ ID NOs: 121 and 122, respectively;

(o) SEQ ID NOs: 133 and 101, respectively; and

(p) SEQ ID NOs: 133 and 102, respectively;

24. The antibody, or antigen binding portion thereof, of embodiment 23,wherein the heavy and light chains comprise the heavy and light chainsselected from the group consisting of (a)-(p).25. The antibody, or antigen binding portion thereof, of embodiment 24,wherein the antibody comprises a heavy chain comprising the amino acidsequence set forth in SEQ ID NO: 135 and a light chain comprising theamino acid sequence set forth in SEQ ID NO: 101.26. The antibody, or antigen binding portion thereof, of embodiment 24,wherein the antibody comprises a heavy chain comprising the amino acidsequence set forth in SEQ ID NO: 135 and a light chain comprising theamino acid sequence set forth in SEQ ID NO: 102.27. The antibody, or antigen binding portion thereof, of any one ofembodiments 23-26, wherein the antibody exhibits any one of thefollowing properties:

(a) inhibits CD73 enzymatic activity;

(b) internalizes into tumor cells or

(c) binds to a conformation epitope comprising amino acids 65-83 and157-172 of human CD73.

28. The antibody, or antigen binding portion thereof, of any one of thepreceding embodiments, wherein the antibody binds to cynomolgus CD73.29. The antibody, or antigen binding portion thereof, of embodiments10⁻²², wherein the antibody comprises an effectorless Fc.30. The antibody, or antigen binding portion thereof, of embodiments 1-7and 9-22, wherein the antibody comprises a modified heavy chain constantregion, comprising a human CH1 domain, a human hinge domain, a human CH2domain, and a human CH3 domain in order from N- to C-terminus.31. The antibody, or antigen binding portion thereof, of embodiment 30,wherein the modified constant region comprises at least 2 domains ofdifferent isotypes selected from the group of isotypes consisting ofIgG1, IgG2, IgG3, and IgG4.32. The antibody, or antigen binding portion thereof, of embodiment 30or 31, wherein the modified constant region comprises a human IgG2 CH1domain and at least one of the CH2, CH3, and hinge domains is not anIgG2 isotype.33. The antibody, or antigen binding portion thereof, of embodiment 32,wherein the IgG2 CH1 domain comprises the amino acid sequence

(SEQ ID NO: 124) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV.34. The antibody, or antigen binding portion thereof, of any one ofembodiments 30-33, wherein the modified constant region comprises ahuman IgG2 hinge domain which reduces heterogeneity in the cysteinebinding.35. The antibody, or antigen binding portion thereof, of embodiment 34,wherein the hinge domain comprises amino acid substitution C219,relative to a wildtype human IgG2 hinge domain (SEQ NO 136).36. The antibody, or antigen binding portion thereof, of embodiment 35wherein the hinge domain comprises the amino acid sequenceERKSCVECPPCPAPPVAG (SEQ ID NO: 123).37. The antibody, or antigen binding portion thereof, of any one ofembodiments 30-36, wherein the modified constant region comprises ahuman IgG1 CH2 domain which reduces or eliminates effector functions.38. The antibody, or antigen binding portion thereof, of embodiment 37,wherein the CH2 domain comprises amino acid substitutions A330S andP331S, relative to a wildtype human IgG1 CH2 domain (SEQ ID NO: 137).39. The antibody, or antigen binding portion thereof, of embodiment 38,wherein the CH2 domain comprises the amino acid sequence

(SEQ ID NO: 125) APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSS IEKTISKAK.40. The antibody, or antigen binding portion thereof, of any one ofembodiments 30-39, wherein the modified constant region comprises ahuman IgG1 CH3 domain.41. The antibody, or antigen binding portion thereof, of embodiment 40,wherein the CH3 domain comprises the amino acid sequence

(SEQ ID NO: 128) GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK.42. The antibody, or antigen binding portion thereof, of any one ofembodiments 9-29, wherein the antibody, or antigen binding portionthereof, is a human or humanized antibody.43. The antibody, or antigen binding portion thereof, of embodiments1-8, wherein methionine residues in the CDR regions are replaced withamino acid residues that do not undergo oxidation.44. A bispecific molecule comprising the antibody of any one of thepreceding embodiments linked to a molecule having a second bindingspecificity.45. An immunoconjugate comprising the antibody according to any one ofembodiments 1-43, linked to a second different agent.46. An isolated nucleic acid molecule encoding the heavy and/or lightchain variable region of the antibody, or antigen binding portionthereof, of any one of embodiments 1-43.47. An expression vector comprising the nucleic acid molecule ofembodiment 46.48. A cell transformed with an expression vector of embodiment 47.49. A composition comprising the antibody, or antigen binding portionthereof, bispecific molecule or immunoconjugate, of any one ofembodiments 1-45, and a carrier.50. A kit comprising the antibody, or antigen binding portion thereof,or bispecific molecule, or immunoconjugate of any one of embodiments1-45, and instructions for use.51. A method of preparing an anti-CD73 antibody, or antigen bindingportion thereof, comprising expressing the antibody, or antigen bindingportion thereof, in the cell of embodiment 48 and isolating theantibody, or antigen binding portion thereof, from the cell.52. A method of decreasing adenosine levels in a tumor cell expressingCD73, comprising contacting the cell with the antibody, antigen bindingportion thereof, bispecific molecule or immunoconjugate, of any one ofembodiments 1-45, such that adenosine levels are decreased.53. A method of stimulating a T cell response against a tumor cellexpressing CD73 in a subject in need thereof, comprising administeringan effective amount of an antibody, or antigen binding portion thereof,bispecific molecule or immunoconjugate, of any one of embodiments 1-45,such that a T cell response is stimulated against the tumor cell.54. A method of stimulating an immune response in a subject comprisingadministering the antibody, or antigen binding portion thereof,bispecific molecule or immunoconjugate, of any one of embodiments 1-45to the subject, such that an immune response in the subject isstimulated.55. The method of embodiment 54, wherein the subject has a tumor cellexpressing CD73 and an immune response against the tumor cell isstimulated.56. A method for inhibiting the growth of tumor cells expressing CD73 ina subject comprising administering to the subject the antibody, orantigen binding portion thereof, bispecific molecule or immunoconjugate,of any one of embodiments 1-45, such that growth of the tumor isinhibited in the subject.57. A method of treating cancer comprising administering to a subject inneed thereof a therapeutically effective amount of the antibody, orantigen binding portion thereof, bispecific molecule or immunoconjugate,of any one of embodiments 1-45, to treat the cancer.58. The method of embodiment 57, wherein the cancer is selected from thegroup consisting of: bladder cancer, breast cancer, uterine/cervicalcancer, ovarian cancer, prostate cancer, testicular cancer, esophagealcancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer,colon cancer, kidney cancer, head and neck cancer, lung cancer, stomachcancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer,skin cancer, neoplasm of the central nervous system, lymphoma, leukemia,myeloma, sarcoma, and virus-related cancer.59. The method of embodiment 57 or 58, wherein the cancer is ametastatic cancer, refractory cancer, or recurrent cancer.60. The method of any one of embodiments 53-59, further comprisingadministering one or more additional therapeutic agents.61. The method of embodiment 60, wherein the additional therapeuticagent is an immunopotentiating molecule (e.g., a PD-1 antagonist, CTLA-4antagonist, LAG-3 antagonist), an anti-CD39 antibody or anti-A2ARantibody.62. A method of detecting the presence of human CD73 in a samplecomprising contacting the sample with the antibody, or antigen bindingportion thereof, of any one of embodiments 1-45, under conditions thatallow for formation of a complex between the antibody, or antigenbinding portion thereof, and CD73, and detecting the formation of acomplex.

The present disclosure is further illustrated by the following examples,which should not be construed as further limiting. The contents of allfigures and all references, Genbank sequences, patents and publishedpatent applications cited throughout this application are expresslyincorporated herein by reference. In particular, the disclosures of PCTpublications WO 09/045957, WO 09/073533, WO 09/073546, WO 09/054863 andPCT/US2013/072918, and U.S. Patent Publication No. 2011/0150892 areexpressly incorporated herein by reference.

EXAMPLES Example 1: Generation of Human Anti-CD73 Antibodies

Human anti-human CD73 monoclonal antibodies were generated in Hco7,Hco27, Hco20, Hco12, Hco17, and Hc2 strains of HuMAb® transgenic mice(“HuMAb” is a Trade Mark of Medarex, Inc., Princeton, N.J.) and KM mice(the KM Mouse® strain contains the SC20 transchromosome as described inPCT Publication WO 02/43478). HC2/KCo27 HuMAb mice and KM mice weregenerated as described in U.S. Pat. Nos. 5,770,429 and 5,545,806, theentire disclosures of which are hereby incorporated by reference.

Mice, including various genotypes of transgenic mice (such as, KM, Hco7,Hco27, Hco20, Hco12, Hco17 and Hc2), were immunized with differentimmunization strategies (different antigen, different dose, duration,routes of administration (footpad (fp), intraperitoneal (ip) andsubcutaneous (sc) and adjuvant (CFA/IFA, Ribi and antibody), etc).Fusions from the mice were performed and screened, and antibodies wereidentified from these fusions. Further characterization led to theisolation of antibodies of particular interest, including the antibodiesdesignated as 11F11-1, 11F11-2, 4C3-1, 4C3-2, 4C3-3, 4D4-1, 10D2-1,10D2-2, 11A6-1, 24H2-1, 5F8-1, 5F8-2, 6E11-1, and 7A11-1. Table 7(below) provides the IgG isotype and allotype of the heavy chains, aswell as the type of light chain, for each antibody. Antibodies thatdiffer only in the light chain are represented by a different digitafter the dash. For example, 11F11-1 has the same heavy chain as11F11-2, but 11F11-1 has the light chain VK1, whereas 11F11-2 has thelight chain VK2. Unless specified otherwise, recombinant antibodiesbased on VH regions of the antibodies in the table were made with thepredominant light chain.

TABLE 7 Predominant Light Other Expressed Light Clone Isotype ChainChains 11F11 IgG2 VK2 VK1 4C3 IgG1za VK1 VK2, VK3 4D4 IgG2 VK1 10D2 IgG4VK2 VK1 11A6 IgG1za VK1 24H2 IgG4 VK1 5F8 IgG1za VK1 VK2 6E11 IgG1za VK17A11 IgG1za VK1The amino acid and nucleotide sequences of the full length sequence ofthe heavy and light chains, the VH and VL domains and the CDRs of eachantibody are provided in the Sequence Listing and in Table 35. The VHand VL amino acid sequences are also provided in FIGS. 1A through 17B,and an alignment of the VH and VL amino acid sequences of the variousantibodies is provided in FIG. 35 (CDR sequences are in bold).

Example 2: Amino Acid Substitutions in Variable Regions and IsotypeVariations

The framework region of the VH region of antibody 11F11 was mutated byintroducing one of more of the mutation at the following amino acidresidues (surrounding amino acids are shown and the mutated amino acidis underlined): T25 (framework mutation; . . . RLSCATSGFTF . . . ), L52(CDR2 mutation; . . . WVAVILYDGSN . . . ), G54 (CDR2 mutation; . . .VILYDGSNKYY . . . ) and V94 (framework mutation; . . . AEDTAVYYCAR . . .). The names of the constructs and the substitutions in each of them areset forth in Table 8:

TABLE 8 Ab Name Originating Ab Substitution CD73.3 4C3 V94A CD73.4 11F11T25A CD73.5 T25S CD73.6 T25A, G54S CD73.7 T25S, G54S CD73.8 T25A, L52W,G54S CD73.9 T25S, L52W, G54S CD73.10 T25A, L52W, G54E CD73.11* 4D4 A25,W52, E54 *CD73.11 is 4D4 and contained these amino acid residues asisolated. It is listed in the Table for comparative purposes.

The constant region of antibodies 11F11 and 4D4 was also modified, byswitching it to an IgG2 constant region (CH1, hinge, CH2 and CH3) with aC219S substitution (“IgG2CS”; SEQ ID NO:267), an effectorless IgG1constant region with the substitutions L234A, L235E, G237A, A330S andP331S (“IgG1.1f”; SEQ ID NO:268) or an effectorless IgG1/IgG2 hybridconstant region that contains a CH1 and hinge from IgG2 (with C219S) andCH2 and CH3 of IgG1 (with A330S/P331S) (“IgG2CS-IgG1.1f” or“IgG2C219S-IgG1.1f”; SEQ ID NO:169). The constructs that were made arelisted in Table 9.

TABLE 9 Origi- VH Constant Ab Name nating Ab substitution region Name ofAb CD73.4 11F11 T25A IgG2CS CD73.4-IgG2CS CD73.4 T25A IgG2CS-CD73.4-IgG2CS IgG1.1f IgG1.1f CD73.6 T25A, G54S IgG2CS- CD73.6-IgG2CSIgG1.1f IgG1.1f CD73.8 T25A, L52W, IgG2CS- CD73.8-IgG2CS G54S IgG1.1fIgG1.1f CD73.10 T25A, L52W, IgG2CS- CD73.10-IgG2CS G54E IgG1.1f IgG1.1fCD73.10 T25A, L52W, IgG1.1f CD73.10- IgG1.1f G54E CD73.10 T25A, L52W,IgG2CS CD73.10-IgG2CS G54E CD73.11 4D4 A25, W52, E54 IgG2CSCD73.11-IgG2CSThe amino acid sequence of CD73.4-IgG2CS IgG1.1f is shown in FIG. 18(SEQ ID NO: 189).

Abs CD73.3-CD73.11 were made as follows. Light chain VK2 (SEQ ID NO:102) was used for the antibodies deriving from 11F11 (CD73.4, CD73.6,CD73.8 and CD73.10). The heavy and light chains were expressed inHEK293-6E cells and culture media were harvested 5 days aftertransfection.

Binding of the constructs to human FcγRs was measured via SPR. hCD64 andhCD32a-H131 binding data for IgG1.1 and IgG2 molecules were consistentwith expected values for the different Fcs. IgG1.1f is the most inertFc. IgG2 and IgG2-C219S showed typical FcR binding for IgG2. Asexpected, data for IgG2-C219S-G1.1f suggests significantly weakerbinding than wild type IgG1 or IgG2, but increased binding compared toIgG1.1f. IgG2-C219S-G1.1f had weak hCD32a-H131 binding (K_(D) of 2.3 μM)and the binding affinity to all other FcγRs were less than 5 μM. Bindingaffinity of IgG2-C219S-G1.1f to cyno FcγRs was more than 5 M. SPRanalysis of binding IgG2-C219S-G1.1f to human FcRn showed pH-dependentbinding (strong at pH6, and weak binding with fast dissociation at pH7.4).

The recombinant preparations were found to frequently lack theC-terminal Lys of the heavy chain. For example, 97% of the heavy chainsof Ab CD73.4.IgG2-C219S-G1.1f lacked the C-terminal lysine. Certainpreparations had pyro-Q at the N-terminal Q (glutamine) of the heavychain. For example, 94% of the N-terminal glutamine of the heavy chainof Ab CD73.4.IgG2-C219S-G1.1f was pyro-Q.

Example 3: Binding Characteristics of Anti-CD73 Antibodies A. SurfacePlasmon Resonance (SPR)

CD73 binding kinetics and affinity were studied by surface PlasmonResonance (SPR) using a Biacore T100 instrument (GE Healthcare) at 25°C.

One experimental format tested the binding of the N-terminal domain ofhCD73 (consisting of residues 26-336 of human CD73; termed N-hCD73) toantibodies that were captured on immobilized protein A surfaces. Forthese experiments, protein A (Pierce) was immobilized to a density of3000-4000 RU on flow cells 1-4 of a CM5 sensor chip (GE Healthcare)using standard ethyl(dimethylaminopropyl) carbodiimide(EDC)/N-hydroxysuccinimide (NHS) chemistry, with ethanolamine blocking,in a running buffer of 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA,0.005% v/v tween 20. Kinetic experiments were performed by firstcapturing antibodies (5-10 ug/ml) on the protein A surfaces using a 30 scontact time at 10 ul/min, with binding of 600, 200, 66.7, 22.2, 7.4,and 2.5 nM N-hCD73-his, using a 180 s association time and 360 sdissociation time at a flow rate of 30 ul/min. The running buffer forthe kinetic experiments was 10 mM sodium phosphate, 130 mM sodiumchloride, 0.05% tween 20, pH 7.1. The surfaces were regenerated aftereach cycle using two 30 s pulses of 10 mM glycine pH 1.5 at a flow rateof 30 μl/min. Sensogram data was double-referenced and then fitted to a1:1 Langmuir model using Biacore T100 evaluation software v2.0.4, todetermine the association rate constant (ka), the dissociation rateconstant (kd), and the equilibrium dissociation constant (KD).

The results are shown in Table 10. The table compiles data fromdifferent experiments. For antibodies for which two or more sets ofnumbers are shown, each set corresponds to data obtained in a separateexperiment.

TABLE 10 Kinetics of CD73 mAbs binding to N-hCD73- his(hCD73(26-336)His) at 25 C. ° mAb Fc ka (1/Ms) kd (1/s) KD (nM) 11F11IgG2 2.6E+05 4.2E−04 1.6 2.9E+05 1.6E−04 0.56 4C3 IgG1 2.2E+04 2.4E−03110 2.4E+04 2.2E−03 92 4D4 IgG2 8.2E+04 7.7E−04 9.4 7.9E+04 4.9E−04 6.210D2 IgG4 6.1E+05 9.5E−04 1.6 11A6 IgG1 5.5E+04 7.6E−03 140 1H9 IgG13.3E+05 9.3E−04 2.8 24H2 IgG4 2.3E+05 3.2E−03 14 5F8 IgG1 1.5E+056.0E−03 41 6E11 IgG1 5.7E+04 1.4E−03 25 7A11 IgG1 8.8E+05 3.8E−04 0.43CD73.4 IgG1.1f 4.2E+05 3.9E−04 0.92 CD73.4 IgG2-C219S 2.9E+05 1.6E−040.55 2.8E+05 3.3E−04 1.2 2.9E+05 3.7E−04 1.3 3.5E+05 4.4E−04 1.2 CD73.4IgG2-C219S-IgG1.1f 3.1E+05 3.5E−04 1.1 3.3E+05 1.4E−04 0.43 3.1E+051.3E−04 0.42 3.2E+05 1.5E−04 0.47 3.1E+05 4.1E−04 1.4 2.7E+05 3.8E−041.4 3.0E+05 4.1E−04 1.4 3.1E+05 4.2E−04 1.3 3.2E+05 4.3E−04 1.3 2.9E+054.0E−04 1.4 CD73.10 IgG1.1f 2.7E+05 1.3E−03 4.7 CD73.10 IgG2-C219S2.2E+05 1.4E−03 6.2 2.2E+05 1.8E−03 8.3 CD73.10 IgG2-C219S-IgG1.1f2.4E+05 1.4E−03 5.7 2.3E+05 1.60E−03  6.8 CD73.3 IgG1.1f 1.6E+04 3.6E−03220 CD73.11 IgG2-C219S 8.0E+04 2.8E−04 3.5 7.9E+04 5.1E−04 6.5 CD73.6IgG1.1f 3.7E+05 2.5E−04 0.68 CD73.6 IgG2-C219S-IgG1.1f 3.0E+05 2.2E−040.72

The K_(D) in the table is the monovalent K_(D), i.e., K_(D) of bindingof the antibodies to the N-terminal portion of human CD73, which ismonovalent.

The G54S mutation is tolerated and appears to slightly increaseaffinity, while removing the predicted DG isomerization site. The L52Wmutation appears to cause a decrease in affinity of approximately 10fold. The 4D4 variants have unique CDR3 sequences and different kinetics(slower association compared to 11F11 molecules).

The average K_(D) from 10 experiments for CD73.4-IgG2-C219S-IgG1.1f is1.1±0.4 nM. The T25A mutation relative to 11F11 does not impact theaffinity.

The results show that all anti-CD73 antibodies bind to human CD73 withgood affinity and have a slow dissociation rate.

The results of the binding studies indicate that binding activity wasmaintained following introduction of mutations into 11F11, 4C3 or 4D4,or isotype switch, although some antibodies had reduced affinityrelative to the original antibody (i.e., 11F11, 4C3 or 4D4). Inparticular, CD73.10 (T25A,L52W,G54E) has a faster dissociation rate thanCD73.4 (T25A) or CD73.11 (4D4). Comparison of all IgG2 moleculesindicates that 11F1 and CD73.4 (11F11-T25A) have the highest monovalentCD73 affinity (KD=1.1 nM±0.4 nM). CD73.10 (11F11-T25A, L52W, G54E) has˜10-fold lower CD73 affinity than 11F11 or CD73.4. This suggests eitherL52W or G54E or both mutations reduce CD73 affinity when in combinationwith other 11F11 sequences. 4D4 and CD73.11 have affinity comparable toCD73.10 (KD ˜5 nM), but different kinetics. 4C3 epitope is believed toinclude regions of N- and C-domains of CD73, therefore the measured KDfor an isolated N-domain is weak (KD=100-200 nM).

Binding of CD73.4-IgG2-C219S-IgG1.1 f to cyno CD73 was alsoinvestigated. The specificity of CD73.4-IgG2-C219S-IgG1.1 f for bindingcynomolgus monkey CD73 was compared to that of binding to human CD73 bysurface Plasmon resonance (SPR) using a Biacore T100 instrument (GEHealthcare) at 25° C. The full length extracellular domain of eitherhuman CD73 (consisting of residues 27-547 of human CD73 linked to a Histag, termed hCD73-his) or cynomolgus CD73 (consisting of residues 27-547of cynomolgus CD73 linked to a His tag, termed cy-CD73-his) were testedfor binding to antibodies that were captured on immobilized protein Asurfaces. For these experiments, protein A (Pierce) was immobilized to adensity of 3000-4000 RU on flow cells 1-4 of a CM5 sensor chip (GEHealthcare) using standard ethyl(dimethylaminopropyl) carbodiimide(EDC)/N-hydroxysuccinimide (NHS) chemistry, with ethanolamine blocking,in a running buffer of 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA,0.005% v/v tween 20. Experiments were performed by first capturingantibodies (5-10 ug/ml) on the protein A surfaces using a 30 s contacttime at 10 ul/min, with binding of 600, 200, 66.7, 22.2, 7.4, and 2.5 nMhCD73-his or cyno-CD73-his, using a 180 s association time and 360 sdissociation time at a flow rate of 30 ul/min. The running buffer forthese experiments was 10 mM sodium phosphate, 130 mM sodium chloride,0.05% tween 20, pH 7.1. The surfaces were regenerated after each cycleusing two 30 s pulses of 10 mM glycine pH 1.5 at a flow rate of 30μl/min.

The results, which are shown in FIG. 19, indicate thatCD73.4-IgG2-C219S-IgG1.1f binds with similar affinity and kinetics tocyno and human CD73. CD73.4-IgG2-C219S-IgG1.1f binds to full lengthhuman and cyno CD73 dimer with a KD of less than 1 nM. No significantcross-reactivity of CD73.4-IgG2-C219S-IgG1.1f to mouse or rat CD73 wasobserved.

The kinetics and affinity of an isolated Fab fragment from the 11F11antibody was also evaluated by SPR. In these experiments, Fab domainfrom a murine anti-6×His antibody was immobilized on a CM5 sensor chipusing EDC/NHS to a density of ˜3000 RU. Full-length hCD73-his wascaptured to 10 RU density on Fc2 (1 ug/ml hCD73-his), 40 RU density onFc3 (5 ug/ml hCD73-his) and 160 RU density on Fc4 (20 ug/ml hCD73-his),using a 30 s contact time at 10 ul/min. Next, the 11F11 Fab fragment(purified from pepsin-cleaved L-cysteine-reduced 11F11 antibody) wastested for binding at 400, 135, 44.4, 14.8, 4,9, 1.7, 0.55 nM, using 180s association time, 600 s dissociation time at 30 ul/min, in a runningbuffer of 10 mM sodium phosphate, 130 mM sodium chloride, 0.05% tween20, pH 7.1. The surfaces were regenerated after each cycle using two 15s pulses of 10 mM glycine pH 2.0 at a flow rate of 30 μl/min. Sensogramdata was double-referenced and then fitted to a 1:1 Langmuir model usingBiacore T100 evaluation software v2.0.4, to determine the associationrate constant (ka), the dissociation rate constant (kd), and theequilibrium dissociation constant (K_(D)). The results are shown inTable 11 below.

TABLE 11 Kinetics of 11F11-Fab binding to hCD73-his surface at 25 C. °hCD73-his surface ka kd K_(D) density (RU) (1/Ms) (1/s) (nM) 10 1.2E+068.7E−04 0.73 40 1.2E+06 8.7E−04 0.73 160 1.1E+06 8.5E−04 0.77

Thus, the results show that the 11F11 Fab fragment has high affinity forhCD73 (K_(D) ˜0.74 nM).

B. Binding of CD73 Antibodies to CD73 Positive Cells

Titration binding curves were produced with CD73 antibodies on Calu6(CD73 endogenous expressors; human pulmonary adenocarcinoma cell line),DMS114 (CD73 negative; human small cell lung cancer cell line),CHO-cynoCD73 (cynoCD73-transfected) and CHO-K1 (cynoCD73 negative),cells using Alexa Fluor® 647 Goat Anti-Human IgG (H+L) as a secondaryantibody, Invitrogen Cat#A-21445, using the following method: 100000cells were plated in 100 uL PBS+2% FBS per well and blocked for 20 min.Using a U-bottom 96-deep well plate, volumes of antibody and PBS+2% FBSwere combined as dictated by Table 12 below.

TABLE 12 [Stock] [Stain] Vol Ab Vol TM Clone (mg/mL) (mg/ml) (uL) (uL)11F11 3.70 0.020 2.92 537.1 CD73.10-IgG1.1f 1.3 0.020 8.31 531.7CD73.10-IgG2 1 0.020 10.80  529.2 CD73.10-IgG2CS-IgG1.1f 1 0.020 1-/80529.2 CD73.4-IgG2 2.3 0.020 4.70 535.3 CD73.4-IgG2CS-IgG1.1f 2 0.0205.40 534.6 CD73.4-IgG1.1f 2.3 0.020 4.70 535.3

An 8-point serial dilution was performed by diluting a sixth of thevolume (90 uL) into 450 μL PBS+2% FBS. The cell plate was spun down for5 minutes at 1500 rpm. 100 uL of diluted antibody was added per well ofthe plate. 100 uL PBS+2% FBS were added to all other wells. The plateswere stained on ice for 45 min, spun down at 1500 rpm for 5 min andwashed twice in 200 uL PBS+2% FBS per well. Wells that had receivedunconjugated antibody, plus one unstained well per cell line, wereresuspended in 100 uL APC anti-human secondary antibody (20 ug/mL). 100uL PBS+2% FBS was added to all other wells, and stained on ice for 45min. The plates were spun down at 1500 rpm for 5 min and washed in 200uL PBS+2% FBS per well. The plates were washed again, resuspended in 200uL 2% FBS in PBS per well and the samples were run.

The results, which are shown in FIGS. 20A1, 20A2, 20B1, 20B2, 20C1,20C2, 20D1, 20D2, and Table 13, indicate that all the CD73 antibodiesbind to cells that naturally express CD73 (Calu6 cells) and CHO cellstransfected to express cyno CD73, but that the antibodies do not bind tocells that do not express CD73 (DMS114 and CHO-K1). The EC50 of bindingobtained for each antibody are shown in Table 13.

TABLE 13 EC50 nM EC50 nM Antibody Calu6 CHO-cynoCD73 11F11 0.78 0.58CD73.10-IgG1.1f 0.64 0.67 CD73.10-IgG2 0.85 1.24 CD73.10-IgG2CS-IgG1.1f0.85 1.27 CD73.4-IgG2 0.49 0.34 CD73.4-IgG2CS-IgG1.1f 0.53 0.51CD73.4-IgG1.1f 0.43 0.45

The EC50 of binding of CD73.4-IgG2-IgG1.1f to human tumor cell lines was0.5 nM (range of 0.3 to 0.67 nM). The EC50 of binding ofCD73.4-IgG2-IgG1.1f to cyno CD73 transfected CHO cells was 0.3 nM (range0.1 to 0.5 nM).

Binding of CD73.4 antibody to human B and T cells was also determined.Human blood from two donors, D316 and D329, was obtained fromImmunsciences, BMS. Peripheral blood mononuclear cells (PBMC) wereisolated with Lympholyte-H cell separation gradient media. PBMC wereincubated with serially diluted FITC-labeled CD73.4-IgG2,CD73.4-IgG2-IgG1.1f, or CD73.4-IgG1.1f antibodies, and T cells and Bcells were identified with fluorochrome-labeled antibodies to CD3 andCD20. Cells from both donors were pooled for the unstained and FMO(Fluorescence minus one) control samples. The results, which are shownin FIGS. 20E and F and Table 14, indicate that the antibodies bindspecifically to human B and T cells.

TABLE 14 IC50 of binding of CD73 antibodies to B and T cells IC50 (nM)IC50 (nM) B cells T cells D316 mAb-CD73.4-IgG2CS-IgG1.1f 0.1648 0.1829D316 mAb-CD73.4-IgG2 0.1588 0.1799 D316 mAb-CD73.4-IgG1.1f 0.0994 0.1263D329 mAb-CD73.4-IgG2CS-IgG1.1f 0.1454 0.2406 D329 mAb-CD73.4-IgG20.07766 0.1348 D329 mAb-CD73.4-IgG1.1f 0.1356 0.2248

Example 4: Biophysical Characteristics of Anti-CD73 Antibodies

A. Size-Exclusion Chromatography Coupled to an in-Line Multi-Angle LightScattering Detector (SEC-MALS)

The oligomeric state of CD73 mAbs were examined by size-exclusionchromatography coupled to an in-line multi-angle light scatteringdetector (SEC-MALS). Isocratic separations were performed on a ShodexPROTEIN KW-803 column connected to an Prominence Shimadzu UFLC in buffercontaining 200 mM K₂HPO₄, 150 mM NaCl, pH 6.8, containing 0.02% Na azide(0.1 μm filtered) running at 0.5 mL/min. Samples were injected onto thecolumn using a SIL-20AC Prominence Shimadzu autosampler, and data wereobtained from three online detectors connected in series: a ProminenceSPD-20AD diode array UV/vis spectrophotometer followed by a WyattminiDAWN™ TREOS Multi-Angle Light Scattering Detector then a WyattOptilab T-rEX Refractive Index Detector. Data (as shown in Table 15below) were collected and analyzed using Astra (Wyatt) and Labsolutions(Shimadzu) software. The results are shown in Table 15.

B. Differential Scanning Calorimetry (DSC)

The thermal stability of CD73 mAbs were determined using a MicroCalCapillary DSC instrument (GE Healthcare). Antibodies were analyzed at aconcentrations of 0.5-0.75 mg/ml in PBS pH 7.1. To stabilize the DSCinstrument baseline and obtain a consistent thermal history, multiplescans of buffer alone in both the sample and reference cell wererecorded prior to sample analysis. Sample scans contained mAb in thesample cell and PBS pH 7.1 in the reference cell. All scans were runfrom 10-110° C. at a scan rate of 60°/hr using a 5 minute pre-cyclethermostat period and no post-cycle thermostat period. Data (as shown inTable 15 below) were analyzed using MicroCal Origin Cap DSC analysissoftware. The appropriate buffer-buffer blank scans were subtracted fromthe sample-buffer data, and the transition midpoint temperature (Tm)values were determined by fitting the data to a non-2-state model. Theresults are shown in Table 15. Tm1, Tm2 and Tm3 are the Tms fordifferent domains in the antibodies.

TABLE 15 SEC-MALS and DSC MALS Mass SEC SEC SEC (main DSC DSC DSC DSC %% % peak/ Tonset Tm1 Tm2 Tm3 mAb Fc HMW Monomer LMW monomer) (° C.) (°C.) (° C.) (° C.) 7A11 0.5 98.5 0.5 146.3 56.0 64.8 70.2 82.8 6E11 2.197.6 0.1 145.2 55.0 62.3 72.0 83.3 11F11 0.8 99.2 0.0 143.3 64.0 73.378.0 5F8 2.3 97.7 0.0 143.8 59.0 68.7 82.7 4C3 0.9 94.4 4.5 142.7 60.066.9 71.2 82.7 11A6 4.8 94.0 0.0 143.2 61.0 66.0 71.4 82.1 10D2 1.1 98.80.0 141.4 61.0 67.7 77.1 24H2 0.0 100.0 0.0 142.4 62.0 71.7 76.9 79.84D4 3.2 96.8 0.0 144.2 62.0 71.7 77.0 79.9 CD73.4 IgG1.1f 98.2 1.8 140.459 65.5 81.2 CD73.4 IgG2-C219S 60 72.9 77.5 CD73.4 IgG2-C219S- 0.4 99.6141.5 59 68.4 78.3 IgG1.1f CD73.10 IgG1.1f 0.4 99.6 135.9 55 64.2 78.2CD73.10 IgG2-C219S 100 152 61 73.2 77.0 CD73.10 IgG2-C219S- 100 139.5 6170.4 76.5 84.1 IgG1.1f CD73.3 IgG1.1f 0.6 99.4 146.1 56 64.8 75.0 83.4CD73.11 IgG2-C219S 61 73.4 77.9 CD73.6 IgG1.1f 0.2 99.7 0.0 142.0 5864.2 79.7 CD73.6 IgG2-C219S- 0.3 99.7 0.1 142.3 60 70.1 77.4 84.6IgG1.1fThe results show that all antibodies are mostly monomeric and arestable.

Example 5: Inhibition of Enzymatic Activity by Anti-CD73 Abs A.Inhibition of Bead-Bound CD73 Enzymatic Activity

To assess bead-bound CD73 enzyme activity inhibition by anti-CD73antibodies, the following materials and methods were used:

Materials

TM buffer: 25 mM Tris, 5 mM MgCl₂ in water0.5 mM Sodium Phosphate buffer, pH8.0Wash buffer (10 mL 0.5 mM Sodium phosphate, pH8.0; 10 mL 5M NaCl; 34 mLwater; 10 uL Tween-20)Adenosine 5′-monophosphate disodium salt, Sigma Cat#01930-% G, 300 mM inTM bufferAdenosine 5′-triphosphate disodium salt hydrate, Sigma Cat#A6419-1G, 100mM in TM buffer rhCD73, 0.781 mg/mLcyno CD73, Sino Biological Inc Cat#90192-C08HMagnet his-tag beads, Invitrogen Cat#10103D

CellTiter-Glo@ Luminescent Cell Viability Assay, Promega Cat#G7572

mAbO, an unrelated antibody that does not bind CD73

Methods

A 6-point serial dilution of the anti-CD73 antibodies listed in Table 16(max concentration 10 ug/mL) was conducted by combining volumes asdictated in Table 16, and diluting 3-fold (transferring 225 uL into 450uL TM buffer). All antibodies with an IgG2 hinge contained the C219Smutation.

TABLE 16 Vol Vol [Stock] [Stim] Ab TM Clone (mg/mL) (mg/mL) (uL) (uL)mAbO 5.38 0.010 1.25 673.7 F3713.11F11.F3.A4 3.70 0.010 1.82 673.2mAb-CD73.10-Vh-hHC- 1.3 0.010 5.19 669.8 IgG1.1f mAb-CD73.10-Vh-hHC-IgG21 0.010 6.75 668.3 mAb-CD73.10-Vh-hHC-IgG2- 1 0.010 6.75 668.3 IgG1.1fmAb-CD73.4-Vh-hHC-IgG2 2.3 0.010 2.93 672.1 mAb-CD73.4-Vh-hHCIgG2- 20.010 3.38 671.6 IgG1.1f mAb-CD73.4-Vh-IgG1.1f 2.3 0.010 2.93 672.1

Magnet beads (2 ul beads per sample) were washed in 1 mL Sodiumphosphate buffer in a microcentrifuge tube. The beads were pulled downwith the magnet and resuspended in 400 uL TM buffer. For each species ofCD73: In a separate tube, CD73 (75 ng per sample) was combined with TMto bring the volume up to 400 uL. A third tube was prepared for blankbeads (no CD73). The bead suspension was combined with rhCD73 solutionand mixed on a shaker for 5 min at room temperature. The beads werepulled down with a magnet and the beads were washed in 1 mL wash buffer.The beads were pulled down with a magnet and resuspended in TM buffer(40 uL per sample). The beads were transferred to PCR 96-well plates (40uL per well). 200 uL per well of serially diluted CD73 HuMab were addedto plates and mixed well. The plates were incubated for 30 min at roomtemperature. 700 uL each of 400 uM ATP (8×) and 1.2 mM AMP (8×) wereprepared. 650 uL of each were combined to make a 4×AMP/ATP stock mix.The beads were pulled down and washed twice with 200 uL TM buffer perwell. The beads were pulled down again and resuspended in 30 uL TMbuffer. The 30 uL beads were transferred to 96 well black plates. 10 uLof the 4× stock solution of AMP/ATP (final concentration 150 uM AMP/50uM ATP) was added and mixed. Control wells (final concentration 150 uMAMP and/or 50 uM ATP) in 40 uL volume were added. The plates wereincubated for 15 min at 37° C.

The results are shown in FIGS. 21A1, 21A2, 21B1, and 21B2, and Table 17.

TABLE 17 mAB Fc EC50 (nM) 11F11 IgG2 3.98 4C3 IgG1 3.63 4D4 IgG2 5.3110D2 IgG1 6.94 11A6 IgG1 3.12 24H2 IgG1 4.18 5F8 IgG1 5.76 6E11 IgG13.71 7A11 IgG1 2.86 CD73.4 IgG1.1f 3.25 CD73.4 IgG2-C219S 2.72 CD73.4IgG2-C219S-IgG1.1f 2.97 CD73.10 IgG1.1f 4.69 CD73.10 IgG2-C219S 7.54CD73.10 IgG2-C219S-IgG1.1f 4.84

The results of enzymatic inhibition of cyno CD73 are set forth in Table18.

TABLE 18 mAB Fc EC50 (nM) CD73.4 IgG1.1f 7.123 CD73.4 IgG2-C219S 3.658CD73.4 IgG2-C219S-IgG1.1f 4.572 CD73.10 IgG1.1f 10.2 CD73.10 IgG2-C219S8.783 CD73.10 IgG2-C219S-IgG1.1f 9.935

The results show that the antibodies dose dependently inhibit theenzymatic activity of human CD73. CD73.4.IgG2-C219S-IgG1.1f has an EC50of 2.97 (range 2.9 to 3.1 nM) in the recombinant human CD73 enzymeinhibition assay. CD73.4.IgG2-C219S-IgG1.1f has an EC50 of 3.7 (range1.6 to 12.6 nM) in the recombinant cyno CD73 enzyme inhibition assay.Thus, all antibodies to CD73 tested inhibit bead bound human and cynoCD73 enzymatic activity.

B. Inhibition of CD73 Enzymatic Activity in Calu6 Cells

This example describes the assessment of Calu6 (CD73 positive) andDMS-114 (CD73 negative) cells for CD73 dephosphorylation of AMP aftertreatment with anti-CD73 antibodies.

Materials:

CD73 antibodies; see table belowMabO control antibody, 5.38 mg/mLTM buffer: 25 mM Tris, 5 mM MgCl₂ in waterAdenosine 5′-monophosphate disodium salt, Sigma Cat#01930-5G, 300 mM inTM bufferAdenosine 5′-triphosphate disodium salt hydrate, Sigma Cat#A6419-1G, 100mM in TM buffer rhCD73, 0.781 mg/mL

CellTiter-Glo® Luminescent Cell Viability Assay, Promega Cat#G7572Methods:

The antibodies were serially diluted by combining volumes of purifiedantibodies and PBS as dictated by Table 19 below in a U-bottom 96-wellplate. 6-point serial dilutions with the antibodies (max concentration25 ug/mL, 300 uL), and 5-fold dilutions, transferring 60 uL into 240 uLPBS, were performed. All antibodies with an IgG2 hinge contained theC219S mutation.

TABLE 19 Conc Vol Vol Clone (mg/mL) Ab (uL) PBS (uL) mAbO 5.38 1.39298.6 F3713.11F11.F3.A4 3.70 2.03 298.0 mAb-CD73.10-Vh-hHC-IgG1.1f 1.35.77 294.2 mAb-CD73.10-Vh-hHC-IgG2 1 7.50 292.5mAb-CD73.10-Vh-hHC-IgG2-IgG1.1f 1 7.50 292.5 mAb-CD73.4-Vh-hHC-IgG2 2.33.26 296.7 mAb-CD73.4-Vh-hHC-IgG2-IgG1.1f 2 3.75 296.3mAb-CD73.4-Vh-hHC-IgG1.1f 2.3 3.26 296.7

Cells were harvested with Versene and counted. Plates were seeded, spundown at 1500 rpm for 5 min, and resuspended in 100 uL serially dilutedantibody. All other wells were resuspended in 100 uL PBS. Incubation wasat 37° C. for 20 min. A 15 mL 180 uM stock of AMP was prepared in TMbuffer.

Plates were spun down at 1500 rpm for 5 min and washed once with 200 uLPBS/well. Plates were spun down again and resuspended in 100 uL AMP. Allother wells were resuspended in 100 uL TM buffer. The cells wereincubated with AMP for 60 min at 37° C. A 7.5 mL 60 uM stock of ATP inTM buffer was prepared. Plates were spun down at 1500 rpm for 5 min and50 uL of the supernatant was transferred to a black 96-well plate. 50 uLof ATP was added. rhCD73 was added to certain wells at 75 ng per well asa positive control. Wells that did not receive rhCD73 were brought up to100 uL with TM buffer. Final concentration was 90 uM AMP: 30 uM ATP.Incubation was at 37° C. for 15 min. For the CellTiterGlo Assay (whichdetects ATP), 100 uL were added per well and the plate was read.

The results, which are shown in FIGS. 22A1, 22A2, 22B1, 22B2, and Table20, indicate that the anti-CD73 antibodies inhibit dephosphorylation ofAMP (or reduce AMP processing) in the human CD73 positive Calu6 cells,but have no effect in CD73 negative DMS114 cells. The EC50 for blockadeof endogenous cellular CD73 in the human tumor cell line Calu6 ofCD73.4-IgG2S-IgG1.1f antibody is 0.39 nM (range 0.31 to 0.48 nM). Theseexperiments were repeated in NCI-H292 (mucoepidermoid carcinoma cellline) and SK-MEL-24 (human melanoma cell line) cells and the resultswere similar (Table 20).

TABLE 20 EC50 EC50 binding EC50 enz. EC50 Calu6 SKMEL24 EC50 H292 Calu6¹ inhibition ² inhibition ³ inhibition ³ inhibition ³ Antibody (nM) (nM)(nM) (nM) (nM) 11F11 0.78 3.980 0.70 3.15 0.81 4C3 2.00 3.63 3.43 13.294.48 4D4 0.82 5.31 11A6 1.93 3.12 2.21 5F8 11.65 5.76 8.10 110.19 13.467A11 0.35 2.86 0.95 3.72 1.31 24H2 4.18 10D2 6.94 6E11 0.63 3.71 1.543.43 1.34 CD73.4-IgG2CS 0.49 2.72 0.34 CD73.4-IgG1.1f 0.43 3.25 0.37CD73.4-IgG2S-IgG1.1f 0.53 2.97 0.39 CD73.10-IgG2S-IgG1.1f 0.85 4.84 0.77CD73.10-IgG1.1f 0.64 4.69 0.77 CD73.10-IgG2S 0.85 7.54 0.84 ¹ Bindingtitration on Calu6 cells with endogenous CD73 expression. Antibodieswere tested in 2-6 independent experiments, and the average value isindicated. ² Data from section A of this Example. Antibodies were testedin 1-5 independent experiments, and the average value is indicated. ³Inhibition of cellular CD73 activity in indicated cell line. Antibodieswere tested in 2-4 independent experiments, and the average value isindicated.C. Inhibition of CD73 Enzymatic Activity in a Dual Cell Line cAMP AssayHomogenous Time Resolved Fluorescence (HTRF) cAMP Assay

CD73 antibodies were serial diluted with PBS buffer containing 0.2% BSA,and plated 5 μl/well in 384 well white bottom proxiplate (PerkinElmer,Waltham, Mass.). Calu-6 cells were harvested and resuspend in PBScontaining 0.2% BSA, then 5 μl of cells (300 cells/well) were added tothe plate. The cells were incubated with antibodies for 10 minutes at37° C. 5% CO₂ and 95% humidity, followed by the addition of 5 μl/well 80mM AMP. The cells were further incubated with AMP for 30 minutes at 37°C. During this time, HEK293/A2AR cells were harvested and diluted to 0.4million/ml in PBS containing 0.2% BSA. They were added into the assayplate at 5 μl/well and continued to incubate at 37′C for 1 hr. HRTFassays were performed using the homogenous time-resolved fluorescence(HTRF) HiRange cAMP detection kit (Cisbio, Bedford, Mass.) by adding 10μl/well cAMP-conjugated d2 and 10 μl/well europium cryptate conjugatedanti-cAMP antibody in lysis buffer according to the manufacturer'sinstructions. Plates were incubated at room temperature for 60 minutesand Fluorescence Resonance Energy Transfer (FRET) signals (665 and 615nM) were read using an EnVision plate reader (PerkinElmer, Waltham,Mass.). The FRET signal was calculated as the ratio of signal from the665 nm (acceptor) and 615 nm (donor) channels and multiplied by 10.000.IC₅₀ and Ymax were measured. Ymax was determined by comparing to 100 nMdose of 11F11 as internal maximum. All calculations were determined as apercentage of inhibition compared to this control, which was set to100%.

The results, which are shown in Table 21, indicate that the anti-CD73mAbs demonstrated different efficacies and potencies in this cAMP assayusing a cell line co-culture system. All Abs showed some reduction inadenosine production, and the extent of inhibition was similar for mostAbs screened. The greatest inhibition was seen for 11F11, 11A6, 4C3 and5F8.

TABLE 21 Substance IC50 (nM) Ymax APCP 1.29 97 11A6 4.87 84 5F8 13.17 804C3 9.02 80 11F11 0.75 76 7A11 0.95 45

Enzymatic inhibition assays were also conducted with 11F11 Fab andF(ab′)₂. The results, which are shown in FIG. 22C, indicated thatenzymatic inhibition occurred with the F(ab′)₂ fragment, but not withthe Fab fragment. Thus, the Fc region is not required for 11F11enzymatic inhibition, but bivalency is required.

Enzymatic inhibition in Calu6 cells was also determined for CD73.4antibodies comprising various heavy chain constant regions, which areshown in Table 26, using the cAMP assay described above. The results, interms of EC50 and level of inhibition versus background (“S:B”) areprovided in the last two columns of Table 28. The results indicate thatall CD73.4 antibodies inhibit human CD73 enzymatic activity in Calu6cells.

D. Time Course of CD73 Enzymatic Activity Inhibition

Inhibition of enzyme activity was also evaluated in a time course byevaluating adenosine generation by LC/MS/MS. Calu6 cells were incubatedwith 11F11 or 4C3 for 30 minutes, 2 hours or 4 hours, followed byaddition of 50 μM AMP and evaluation of adenosine production by LC/MS/MSusing standard methods.

Mass Spectrometry Conditions (Xevo TO-S):

Instrument: Xevo TQ-S (with Waters 2777C)

Tune = CD73_adenosine_MRM_tune2.ipr Ionization: (+) ESI Desolvation Temp(° C.): 500 Capillary (kV): 0.9 Desolvation Gas (L/Hr): 1000 Cone (V):see below Cone Gas (L/Hr): 150 Source Offset V): 50 Nebuliser (Bar): 7.0LM Resolution 1: 2.81 HM Resolution 1: 15.00 LM Resolution 2: 2.93 HMResolution 2: 15.00 Ion Energy 1: 0.4 Ion Energy 2: 0.9 Collision GasFlow (mL/min): 0.15 Collision: see below Sample diverted to waste forfirst 0.5 min Waters Xevo TQ-S Serial number: WAA021

The results, which are shown in FIG. 22D, indicate that incubation timedoes make a difference at the 30 min time point and that inhibition by11F11 occurs faster than that by 4C3. Though both antibodies achievedequal inhibition at later timepoints, the 11F11 antibody more rapidlyinhibits CD73 enzymatic activity in cells.

Example 6: Antibody Mediated Internalization of CD73

The anti-CD73 antibody mediated internalization of CD73 was measured intwo different assays.

A. High-Content Internalization Assay (2 Hour Fixed Time Assay)

The anti-CD73 antibodies were used to test anti-CD73 antibody dependentCD73 internalization in Calu6 cells by assessing cellular expressionafter 2 hours of antibody incubation. Cells (2,000 cells/well) in 20 μlof complete medium (Gibco RPMI Media 1640 with 10% heat inactivatedfetal bovine serum) were plated in 384 BD Falcon plate and grownovernight at 37° C. 5% CO₂ and 95% humidity. Anti-CD73 antibodies wereserially diluted with PBS buffer containing 0.2% BSA, and added at 5μl/well into the cell plate. The cells were incubated with antibodiesfor 2 hours at 37° C. 5% CO₂ and 95% humidity, followed by washing oncewith PBS buffer. Formaldehyde (final 4% in PBS) was then added into cellplate at 20 ul/well, and the plate was incubated at room temperature for10 minutes Afterwards, all liquid was aspirated and cells were washedonce with 30 ul PBS. Detection antibody (2.5 μg/well of anti-CD73 AbCD73.10.IgG2C219S) was added at 15 μg/well into the fixed cell plate.The cells were incubated at 4° C. overnight. On the next day, the platewas washed twice with PBS buffer, followed by adding secondary antibodycontaining Alexa-488 goat anti human and DAPI, stained for 1 hour atroom temperature. After 3 washes in PBS buffer, the plate was imaged onArrayscan Vti (Cellomics, Pittsburgh. Pa.). IC₅₀ and Ymax were measured.Ymax was determined by comparing to 100 nM dose of 11F11 as internalmaximum. All calculations were determined as a percentage ofinternalization compared to this control, which was set to 100%.

The results are provided in Table 22.

TABLE 22 mAb Constant region Epitope bin EC50 (nM) Ymax 11F11 IgG2 10.58 98 4C3 IgG1 2 ND NA 4D4 IgG2 1 0.38 104  10D2 IgG1 1 ND 29 11A6IgG1 1 ND NA 24H2 IgG1 1 8.2  51 5F8 IgG1 2 ND NA 6E11 IgG1 1 ND NA 7A11IgG1 1 2.59 50 CD73.4 IgG2-C219S-IgG1.1f 1 1.2  97 CD73.10 IgG1.1f 16.18 64 CD73.10 IgG2-C219S 1 0.67 99 CD73.10 IgG2-C219S-IgG1.1f 1 0.8799 ND = Not Detected NA = Not Applicable

Thus, the results indicate that the EC50 of CD73 internalizationmediated by CD73.4.IgG2-C219S-IgG1.1f in the human CD73 expressing cellline Calu6 was 1.2 nM, and that the maximal level of internalization inthe cell line was 97.5%.

Internalization assays were also conducted with 11F11 Fab and F(ab′)₂.The results, which are shown in FIG. 22C, indicate that internalizationoccurred with the F(ab′)₂ fragment, but not with the Fab fragment. Thus,the Fc region is not required for 11F11 internalization.

Kinetic internalization studies were performed to assess the rate ofinternalization. Cells (2,000 cells/well) in 20 μl of complete medium(Gibco RPMI Media 1640 with 10% heat inactivated fetal bovine serum)were plated in 384 BD Falcon plate and grown overnight at 37° C. 5% CO₂and 95% humidity. CD73 antibodies were diluted with PBS buffercontaining 0.2% BSA to 10 μg/ml and added 5 μl/well into the cell plate.The cells were incubated with antibodies for a 0-2 hour time course at37° C., followed by washing once with PBS buffer. The cells weresubsequently fixed with formaldehyde (final 4% in PBS) at roomtemperature for 10 minutes, and then washed once with 30 ul PBSDetection antibody (2.5 μg/well anti-CD73 Abs CD73.10.IgG2C219S) werediluted with PBS buffer containing 0.2% BSA, and added 15 μl/well intothe fixed cell plate. The plate was incubated at 4° C. for overnight. Onthe next day, after 3 washes in PBS buffer, Secondary antibodyAlexa488-goat anti human with DAPI were added. The cells were stainedfor 60 minutes at room temperature, after 3 washes, images were acquiredusing Arrayscan Vti (Cellomics, Pittsburgh, Pa.). The results areprovided in FIGS. 23A-23D and Tables 23 and 24. The values in Table 24derive from the data shown in FIGS. 23A-D.

TABLE 23 CD73.10.IgG1.1f 11F11(IgG2) T_(1/2) 6E11 1T_(1/2) T_(1/2) Cellline Cell type (min) (min) (min) Calu6 Human pulmonary 3.9 60.9 14.4adenocarcinoma HCC44 Non-small cell lung 3.3 27.9 23.5 carcinoma H2030Non-small cell lung 3.3 40.3 18.3 carcinoma H647 Non-small cell lung45.7 N/A N/A carcinoma H2228 Non-small cell lung 10.9 36.5 35.7carcinoma HCC15 Non-small cell lung 2.2 84.4 37.9 carcinoma SKLU1 Lungadenocarcinoma 6.8 18.0 17.2 SKMES1 Melanoma 2.2 62.8 32.3 SW900Squamous cell lung 10.3 94.9 43.4 carcinoma

TABLE 24 T_(1/2) and % internalization of CD73 antibodies in 4 humancell lines H228 cells HCC15 cells Calu6 cells H2030 cells % % % %T_(1/2) internal- T_(1/2) internal- T_(1/2) internal- T_(1/2) internal-min ization min ization min ization min ization CD73.11-IgG2CS — — — —4.1 89 4.6 85 CD73.10-IgG2CS 9.7 93 2.6 91 3.0 91 3.3 85 CD73.10-IgG2CS-9.4 92 3.0 91 3.1 91 4.3 87 IgG1.1f CD73.4-IgG2CS 13.8 94 3.1 94 6.5 883.7 89 CD73.10-IgG1.1f 35.7 33 37.9 71 14.4 63 18.3 67 CD73.3-IgG1.1f16.5 −47 >240 38 111.4 79 >120 27 11F11 10.9 96 2.2 94 3.9 87 3.3 90 4C37.6 −48 141.5 28 0.6 −6 >120 -34 6E11 36.5 13 84.4 64 107.4 68 40.32 51

The results indicate that the bin 1 antibodies (11F11 and itsderivatives CD73.4 and CD73.10) showed good internalization EC₅₀ andmaximal values (97.5%), although some antibodies were more internalizedthan others. 11F11 was the most active and internalized within minutes,reaching a plateau in 30 minutes, whereas 6E11 (also a bin 1 antibody,IgG) internalized more slowly, reaching a plateau at about 1 hr (FIGS.23A-D). The bin 2 antibodies (5F8 and 4C3) did not internalizesignificantly. In addition, the presence of IgG2 hinge and CH1 domainenhanced the speed and extent of internalization. This trend wasobserved in several cell lines (FIGS. 23A-D and Table 24).

B. Internalization Measured by Flow Cytometry

Anti-CD73 antibody mediated internalization of CD73 was also tested byflow cytometry. Indicated cells were incubated with 10 μg/mL of theindicated antibody for 30 minutes on ice, washed several times, andtransferred to 37° C. for the indicated time. Cells were harvested atthe same time after the indicated incubation time. Cells were stainedwith primary antibody again (same antibody used for initial incubation)followed by anti-human secondary antibody. Cells were then assayed forexpression of CD73 by flow cytometry.

The results, which are shown in FIG. 23E and Table 25, are consistentwith those obtained in the internalization assays described above, andindicate that, all antibodies with IgG2 hinge and CH1 induced rapid andcomplete internalization. The CD73 levels remained low after 22 hourspost wash-out, indicating that internalization is durable.

Similar results, shown in FIG. 23F and Table 25, were obtained in theNCI-H292 cell line, in which antibody was maintained in culture duringthe incubation time (no wash-out). Again, these data indicate rapid andsignificant internalization and maintenance of downregulation ofendogenous CD73.

Internalization assays were also conducted with the human SNU-C1 (coloncancer cell line) and NCI-H1437 (non-small cell lung carcinoma cellline) cells. The results, which are shown in FIGS. 23I and J and Table25, also indicate rapid internalization with a maximal level reachedwithin 5 hours and a maximal level of internalization of about 50% forCD73.4.IgG2-C219S-IgG1.1f in SNU-C1 and 60% for NCI-H1437 cells. FIGS.23G and H show similar kinetics of internalization ofCD73.4.IgG2-C219S-IgG1.1f in Calu6 and NCI-H292 cells. For graphs, whichshow % of CD73 internalized, this number was obtained as follows:

${\% \mspace{14mu} {CD}\; 73\mspace{14mu} {internalized}} = {100 - \left( {\frac{\left( {{MFI}_{t = x} - {MFI}_{background}} \right.}{{MFI}_{t = 0} - {MFI}_{background}} \times 100} \right)}$

where for each antibody, MFI_(t=x) is the MFI at a given timepoint andMFI_(t=0) is maximal fluorescence at t=0, and MFI_(background) is theMFI of the secondary Ab only.

TABLE 25 EC_(50S) of antibody mediated CD73 internalization in severalcell lines SNU-C1 NCI-H1437 Calu6 NCI-H292 SNU-C1 (no wash) NCI-H1437(no wash) Ymax T_(1/2) Ymax T_(1/2) Ymax T_(1/2) Ymax T_(1/2) YmaxT_(1/2) Ymax T_(1/2) (%) (hr) (%) (hr) (%) (hr) (%) (hr) (%) (hr) (%)(hr) mAb- 76.8 0.5661 77.64 0.2633 48.96 0.4954 38.39 1.025 63.12 0.316462.78 0.3418 CD73.4- IgG2- IgG1.1f mAb- 75.59 0.6003 78.42 0.2766 — — —— — — — — CD73.4- IgG2 mAb- 44.99 1.737 51.49 0.2087 30.58 0.9915 33.162.33 49.76 0.4915 49.95 0.5384 CD73.4- IgG1.1f

Additional internalization assays were conducted in Calu6 and H292 cellsto further discriminate the role of isotype on internalization. Theinternalization assays were conducted as described above (protocolwithout the wash-out step of the antibodies), and the antibodies ofvarying hybrid isotypes shown in Table 26 were maintained in culture at10 g/mL during the incubation time. For the flow cytometry experiments,the method of Example 6B was adapted to high throughput analysis in 96well plates (as opposed to 48 well plates) and with 50,000 cells perwell.

TABLE 26 Constant regions tested with the variable regions of CD73.4:SEQ ID NO of constant Constructs region Description IgG1f 267 wild typeIgG1f IgG1.1f 272 standard inert IgG1.1f IgG2.3 268 IgG2 A-form (C219S)IgG2.5 271 IgG2 B-form (C131S) IgG2.3G1-KH 270 CH1, upper hinge andlower hinge/upper CH2 of IgG2.3, all else IgG1f IgG2.5G1-KH 279 CH1,upper hinge and lower hinge/upper CH2 of IgG2.5, all else IgG1fIgG2.3G1-AY 269 CH1 and upper hinge of IgG2.3, all else IgG1fIgG2.5G1-AY 278 CH1 and upper hinge of IgG2.5, all else IgG1fIgG1-G2.3G1-KH 282 CH1 of IgG1, upper hinge and lower hinge/ upper CH2of IgG2.3, all else IgG1f IgG1-G2.3G1-AY 281 CH1 of IgG1, upper hinge ofIgG2.3, all else IgG1f IgG2.3G1.1f-KH 273 CH1, upper hinge and lowerhinge/upper CH2 of IgG2.3, all else IgG1.1f IgG2.5G1.1f-KH 277 CH1,upper hinge and lower hinge/upper CH2 of IgG2.5, all else IgG1.1fIgG1-deltaTHT 274 IgG1 with THT sequence removed from hingeIgG2.3-plusTHT 275 IgG2.3 with THT sequence (from IgG1) added into hingeIgG2.5-plusTHT 280 IgG2.5 with THT sequence (from IgG1) added into hingeIgG2.3-plusGGG 276 IgG2.3 with flexible GGG sequence added into hinge

FcγR binding was shown to be as expected for each construct, i.e., FcγRbinding is driven by lower hinge/CH2 region.

The results are shown in FIGS. 23K, L, M and in Table 27 and 28. Datashown in Table 27 were generated using the same protocol described inExample 6B. Data shown in Table 28 were generated using the sameprotocol described in Example 6A.

TABLE 27 Ymax and T_(1/2) of antibody mediated CD73 internalization inCalu6 and NCI-292 cells Calu6 NCI-H292 Ymax Ymax (%) T_(1/2) (hr) (%)T_(1/2) (hr) mAb-CD73.4-IgG1f/LC- 55.72 0.8452 73.05 0.5014 11F11-Vk2mAb-CD73.4-IgG2.3G1-AY- 85.07 0.3326 90.25 0.272 pTT5-SPmAb-CD73.4-IgG2.3G1-KH 81.62 0.3962 91.61 0.2801mAb-CD73.4-G1-G2.3-G1-AY 72.7 0.4229 84.51 0.3083mAb-CD73.4-IgG1-deltaTHT 69.27 0.5652 83.63 0.3441mAb-CD73.4-G1-G2.3-G1-KH 65.67 0.5674 83.29 0.343mAb-CD73.4-IgG2.3-plusTHT 81.19 0.3551 91.41 0.2935mAb-CD73.4-IgG2.3-plusGGG 81.72 0.3355 91.6 0.2712 mAb-CD73.4-IgG2.578.98 0.3485 89.56 0.3057 mAb-CD73.4-IgG2.5G1.1f-KH 79.63 0.3527 90.860.2993 mAb-CD73.4-IgG2.5G1-AY 81.91 0.2901 91.3 0.2452mAb-CD73.4-IgG2.5G1-KH 76 0.2837 90.75 0.256 mAb-CD73.4-IgG2.5plusTHT/LC80.15 0.2869 89.6 0.2565 mAb-CD73.4-IgG2-C219S/LC 82.35 0.3725 88.910.2866 mAb-CD73.4-IgG2-C219S/LC 82.54 0.3639 87.66 0.2845mAb-CD73.4-IgG1.1f + K/LC 57.07 1.519 70.4 0.4969mAb-CD73.4-IgG2CS-IgG1.1f 80.98 0.3508 90.35 0.2764

TABLE 28 Internalization and enzyme inhibition characteristics of CD73.4with various constant regions in Calu6 cells Internalization CD73Inhibition # CD73_mAb_Clones Max Speed EC50(nM) S:B 1CD73.4-IgG1f/LC-11F11-Vk2 + + 2.01 2 2CD73.4-Vh-hHC-IgG2.3G1-AY-pTT5-SP5 ++++ ++++ 2.37 56 3CD73.4-Vh-hHC-IgG2.3G1-KH ++++ +++ 1.70 52 4 CD73.4-Vh-hHC-G1-G2.3-G1-AY++ ++ 0.38 6 5 CD73.4-Vh-hHC-G1-G2.3-G1-KH ++ ++ 0.63 3 6CD73.4-Vh-hHC-IgG1-deltaTHT ++ +++ 0.31 6 7 CD73.4-Vh-hHC-IgG2.3-plusTHT++++ ++++ 1.54 33 8 CD73.4-Vh-hHC-IgG2.3-plusGGG ++++ ++++ 1.26 26 9CD73.4-Vh-hHC-IgG2.5 ++++ ++++ 2.17 51 10 CD73.4-Vh-hHC-IgG2.5G1.1f-KH++ ++++ 0.87 45 11 CD73.4-Vh-hHC-IgG2.5G1-AY +++ ++++ 0.43 92 12CD73.4-Vh-hHC-IgG2.5G1-KH +++ ++++ 0.44 42 13CD73.4-Vh-hHC-IgG2.5plusTHT/LC ++++ ++++ 0.90 44 14CD73.4-Vh-hHC-IgG2-C219S/LC ++++ ++++ 1.56 28 15CD73.4-Vh-hHC-IgG2-C219S/LC ++++ ++++ 1.78 41 16 CD73.4-Vh-hHC-IgG1.1f +K/LC + + 0.70 2 17 CD73.4-Vh-hHC-IgG2C219S-IgG1.1f ++++ ++++ 1.28 12

FIGS. 23K, L and M and Tables 27 and 28 indicate that antibodies havinga hinge and CH1 domain of the IgG2 isotype are most efficient at drivinginternalization of CD73, whereas the antibodies that have an IgG1 hingeand CH1 domain correspond to the lower curves in the figure, i.e., lowerextent of internalization. In addition, antibodies with only the hingefrom IgG2 have an increased internalization compared to a human IgG1hinge. Thus, antibodies having a hinge and CH1 domain of the IgG2isotype have superior internalization characteristics relative to theantibodies with an IgG1 isotype.

Thus, anti-CD73 antibody mAb-CD73.4-IgG2CS-IgG1.1f induced rapidinternalization dependent on cell line tested. The T1/2 forinternalization ranged from minutes to under an hour. Most cell linestested had a T1/2 under 10 minutes. A nearly complete internalizationwas induced for some cell lines and all tested had at least a 50%reduction in surface CD73 expression which typically reached maximallevels by 5 hours, much shorter in some cases.

The SEC-MALS and DLS data demonstrate that larger complexes are formedbetween hCD73-his and mAbs containing an IgG2 hinge and CH1 region(IgG2-C219S or IgG2-C219S-IgG1.1f), compared to those containing theIgG1 hinge and CH1 region (IgG1.1f).

Example: 7: CD73 Enzymatic Inhibition in Tumors in Xenograft AnimalModels

Mice bearing subcutaneous human Calu6 tumors were treated withCD73.10-IgG1.1, CD73.10-IgG2CS, or CD73.10-IgG2CS-IgG1.1 after 7 days ofgrowth. Antibodies were dosed at 10 mg/kg IP. Tumors were harvested atdays 1, 2, 3 and 7 after antibody administration, embeded in OCT andsnap frozen in chilled isopentane. OCT embeded tumors were cut in 5-6 μmsections and allowed to dry over night at RT. Tumor sections were fixedfor 2.5 min with a 1:1 mixture of cold 10% phosphate-buffered formalinand acetone then preincubated for 1 hour at RT in 50 mM Tris-maleatebuffer, pH 7.4 containing 2 mM CaCl2) and 0.25 M sucrose. After 1 hourthe preincubation buffer was removed and was replaced with the samebuffer supplemented with 5 mM MnCl₂, 2 mM Pb(NO3)2, 2.5% Dextran T200,2.5 mM levamisole, and 1 mM AMP. The enzymatic reaction was carried outfor 1 h at 37° C. After a rinse with DI water, sections were incubatedfor exactly 1 min with 1% (NH₄)₂S followed by a quick rinse in DI water.Sections were counterstained with haematoxylin, dehydrated and mountedwith a xylene based mounting medium. A brown color indicates thepresence of active CD73, whereas the lack of brown color indicates thatCD73 enzymatic activity was inhibited by the antibody.

The results indicate that CD73.10-IgG1.1, CD73.10-IgG2CS, andCD73.10-IgG2CS-IgG1.1 inhibit CD73 enzymatic activity in vivo.Representative stained sections of the tumors from mice treated with theCD73.10-IgG2CS-IgG1.1 antibody are shown in FIGS. 24 A-E. The stainedsections of tumors from mice treated with the other two antibodies weresimilar. The extent of inhibition of CD73 correlated with serum levelsof antibody. Thus the slightly higher level of CD73 activity observed inthe Day 3 example correlated with a lower serum level of antibody thanthe Day 7 example.

A similar experiment to that described above was conducted on micebearing subcutaneous human SNUC1 colon adenocarcinoma-derived xenografttumors and treated with the anti-CD73 antibody CD73.4IgG2CS-IgG1.1f.Mice with SNUC1 tumors were treated with CD73.4IgG2CS-IgG1.1f at 1, 3and 10 mg/kg IP on day 0. Tumors were harvested at 24h, 48h, 72h, 96hand 168h after dosing. The CD73 enzymatic inhibition assay was performedas described above. The quantification of brown staining was performedwith Image Pro Premier software (Media Cybernetics).

The results, which are presented in the graph in FIG. 24F, show thatCD73 activity is significantly reduced animals dosed with the anti-CD73antibody when compared with control antibody-treated mice, indicatingstrong CD73 enzyme inhibition by the antibody at all threeconcentrations. Thus, the anti-CD73 antibody CD73.4CS-IgG1.1fefficiently inhibits CD73 enzymatic activity in vivo. The kinetics ofCD73 inhibition by the anti-CD73 antibodies was also determined in the4T1 syngeneic tumor model. TY/23 (rat anti-mouse CD73 antibody) or ratIgG control (10 mg/kg) was injected on day 7 post 4T1 tumor cellinjection. Tumor, spleen, whole blood and serum were collected on days1, 2, 3, 6 and 7 after Ab treatment. Inhibition of CD73 activity wasmeasured as described above in sections from the indicated day.Representative tumor sections are shown in FIGS. 25 A and B. The dataindicate that TY/23 inhibits CD73 activity in vivo.

Example 8: Epitope Binning and Flow Cytometry Based Cross-Blocking

Epitope binning studies were performed by Biolayer Interferometry (BLI)using an Octet RED instrument (Pall Fortebio) at 25° C. For thesestudies, 20-30 ug/ml hCD73-his was captured on anti-penta-his sensorsusing a 90-180s loading phase. Antibody competition was evaluated byallowing a given antibody (mAb1) to bind to the hCD73-his surfaces for180s, followed by the immediate exposure to a second antibody solution(mAb2) for 180s. The binding signal for mAb2 after pre-binding of mAb1was compared to that of mAb2 in the absence of competition, to determineif mAb1 and mAb2 compete for binding to the hCD73-his surfaces. Theseexperiments were performed for numerous mAb pairs in both orders (mAb1then mAb2 and mAb2 then mAb1) to establish the competition profiles andepitope bins (as summarized in Table 29 below).

As shown in Table 29, the epitope binning analysis revealed 2 epitopebins.

TABLE 29 Antibody Bin 1 Bin 2 7A11 X 6E11 X 11F11 X 5F8 X 4C3 X 11A6 X

The antibodies were also subjected to flow cytometry basedcross-blocking. The experiment was conducted as follows using one set oflabeled fluorescently labeled antibody and a second set of unlabeledantibody: 100000 NCI-H292 cells were seeded per well. The plate was spundown and the cells were resuspended in 100 uL 2% FBS in PBS per well.The cells were blocked on ice for 20 min. Unlabeled antibody, asindicated, in 2% FBS in PBS was added to each well. The plate was spundown and the cells were resuspended in 100 uL per well of diluted,labeled antibody (10 ug/mL), i.e., either 4C3 or 11F11, conjugated toFITC. 6 wells of cells were incubated without antibody, and wereresuspended in 100 uL 2% FBS in PBS only (for controls). The cells werethen incubated on ice for 30 min. The cells were washed twice with 2%FBS in PBS and the samples were resuspended in 140 uL 2% FBS in PBS, andanalyzed on a FacsCalibur flow cytometer (Becton Dickinson).

The results of the flow cytometry-based cross-blocking, which are shownin FIGS. 26A and B, confirm the SPR epitope binning data set forthabove. For example, 7A11 competes with 11F11, but 4C3 does not.

Example 9: Epitope Mapping by HDX

This Example describes the use of HDX-MS for the identification of theepitope on human CD73 to which CD73.4-IgG2CS-IgG1.1f.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) method probesprotein conformation and conformational dynamics in solution bymonitoring the rate and extent of deuterium exchange of protein backboneamide hydrogen atoms (except proline). The exchange level of HDX dependson protein solvent accessibility and hydrogen bonds, and the massincrease of the protein upon HDX can be precisely measured by MS. Whenthis technique is paired with enzymatic digestion, structure features atthe peptide level can be resolved, enabling differentiation of surfaceexposed peptides from those folded inside. In epitope mappingexperiments, the deuterium labeling and subsequent quenching experimentsare performed in parallel for antigen and antigen/mAb complex, followedby online pepsin digestion, peptide separation, and MS analysis.

Prior to epitope mapping of CD73.4-IgG2-CS-IgG1.1f in CD73 by HDX-MS,non-deuteriated experiments were performed to generate a list of commonpeptic peptides for recombinant human full length ECD dimeric CD73 (12μM) and protein complex of recombinant human CD73 and CD73 mAb (1:1molar ratio, 12 μM for CD73 mAb), achieving a sequence coverage of 88%for full length ECD CD73. In the HDX-MS experiment, 5 μL of CD73 (SEQ IDNO: 99) or CD73 with CD73.4-IgG2-CS-IgG1.1f mAb was diluted into 55 μLof D₂O buffer (10 mM phosphate buffer, D₂O, pD 7.0) to start thelabeling reactions at room temperature. The CD73 protein used wasglycosylated full length dimeric hCD73 having SEQ ID NO: 99, also shownbelow). The reactions were carried out for different periods of time: 20sec, 1 min, 10 min and 240 min. By the end of each labeling reactionperiod, the reaction was quenched by adding quenching buffer (100 mMphosphate buffer with 4M GdnCl and 0.4M TCEP, pH 2.5, 1:1, v/v) and 50μL of quenched sample was injected into Waters HDX-MS system foranalysis. The deuterium uptake levels of common peptic peptides weremonitored in the absence/presence of CD73 mAb.

The CD73 protein used had the amino acid sequence having SEQ ID NO: 99.

HDX-MS measurements on CD73 mAb in CD73 indicate thatCD73.4-IgG2-CS-IgG1.1f mAb recognizes a discontinuous epitope comprisedof two peptide regions in the N-terminal region of CD73:

Peptide region 1 (65-83): FTKVQQIRRAEPNVLLLDA (SEQ ID NO: 96)Peptide region 2 (157-172): LYLPYKVLPVGDEVVG (SEQ ID NO: 97)

A three-dimensional view of the interaction (FIG. 27B) shows that thesetwo regions are geometrically close. A detailed map of the interactionis shown in FIG. 27A.

Example 10: Crystal Structure of 11F11 Binding to CD73

This Example describes the crystal structure of a Fab′ of 11F11 bound toCD73(26-336)His.

CD73(26-336)His was purified from transiently transfected HEK-293 Ecells using standard protocols, and used as such, or was deglycosoylatedby PNGase F treatment, and concentrated to 1.2 mg/ml. Antibody 11F11Fab′ was prepared by Pepsin digestion of 11F11 using standard protocols,and concentrated to 1.1 mg/ml.

The complex was formed by incubating equal volumes of deglycosylatedhCD73(26-336)His and the 11F11 Fab′ overnight at 4° C., purified byusing GE Superdex 200 26/60 column, and concentrated to 9.5 mg/ml usinga 10 k MWCO spin concentrator.

The crystals were grown in sitting drops, vapor diffusion experimentsand the drop was 0.25 uL protein mixed with 0.25 uL reservoir solution.Over 7100 crystallization experiments were set up. Initial crystal leadswere small about 10 μm. Optimized crystals were 200-300 μm in size.Crystallization optimization included screening: additives, detergents,precipitants, pH, temperature, and buffer type. The conditions thatallowed crystal formation were as follows: the reservoir solutionconsisted of 34% Polypropylene Glycol P400, 0.1 M Na/K PO4 pH 6.5, and15 μM CYMAL-7; crystallization experiments setup at room temperature andthen placed at 4° C. to incubate; and incubation at 4° C. for 7 days.Crystal formation was only observed with the glycosylated CD73 protein.

The crystals were harvested directly from the crystallization drop andplaced directly into liquid N2. Over 100 crystals were screened fordiffraction in-house.

Data were collected using a small beam, very little attenuation, andhelical data collection on SER-CAT beamline 22ID with the RayonixMX-33HS high speed CCD detector. Data sets were collected at 4.1 Å, 3.8Å, 3.5 Å, and finally at 3.05 Å. The data, processed and scaled usingroutine HKL2000 (Otwinowski Z., Minor W., Methods in Enzymology 276,307-326 (1997)), was 96% complete to 3.04 Å resolution.

A BLAST (Altschul et al. (1990) “Basic local alignment search tool.” J.Mol. Biol. 215:403-410) search was used to find the closest model forthe CD73 N-terminal domain and the Fc and Fv domains in the RCSB ProteinData Bank to be used in a molecular replacement search: CD73 model wasfrom PDB entry 4H1S (Heuts et al. Chembiochem. 2012 Nov. 5;13(16):2384-91).

These were used as the starting model in the PHASER (McCoy et al. J.Appl. Cryst. (2007). 40, 658-674) molecular replacement search. The CD73search found 5 molecules in the asymmetric unit. Keeping the CD73 fixed,a search with the heavy chain search model found 2 molecules in theasymmetric unit. Keeping the CD73s and heavy chains fixed, a thirdPHASER search with the light chain also found 2 molecules in theasymmetric unit. A composite model of five complete complexes was madefrom the partial solutions by overlaying the five CD73s and matching upthe heavy and light chains. This was used as the starting model for aBUSTER (Bricogne et al. (2011) BUSTER version 2.11.6. Cambridge, UnitedKingdom: Global Phasing Ltd) refinement.

The model has been refitted and the amino acids changed to reflect the11F11 sequences. The model underwent extensive manual model-building andrefinement. A total of five BUSTER refinement cycles were run tocomplete the refinement. The final R-factor is 20.59% (R-free=24.58%)for the 27,484 protein atoms and 24 solvent molecules.

Representations of the crystal structure of the complex are set forth inFIGS. 28A-D.

The crystal structure shows that all but one of the interactions arefrom residues in the CDR regions, and that most of the interactions arefrom the VH domain with two additional interactions from the VL domain(FIG. 28A). The interacting residues of human CD73 and 11F11 Fab′ areshown in Table 30.

TABLE 30 11F11 Heavy Chain 11F11 Light Chain Dis- Dis- CD73 ResidueInteraction Residue tance (Å) Residue tance (Å) Phe-65 VDW Ser-30 4.0Ser-31 3.5 Trp-32 3.8 Gln-69 VDW Trp-32 3.9 Arg-73 VDW Ser-53 3.8Asn-106 VDW Tyr-100 3.6 Ala-107 Trp-32 3.7 Arg-109 H-Bond Pro-100A 2.8Tyr-91 3.0 VDW Tyr-100 3.4 Trp-32 3.5 Asn-92 3.5 Tyr-100 H-Bond Tyr-1003.0 Lys-136 VDW Trp-99 3.3 Tyr-100 3.6 Phe-137 VDW Trp-99 3.6 Tyr-1003.3 Pro-138 VDW Trp-99 3.4 Lys-162 Salt Link Asp-53 2.8 VDW Tyr-52A 3.2Trp-99 3.8 Leu-164 VDW Tyr-52A 3.6 Pro-165 VDW Asn-31 3.2 Tyr-52A 3.6Ser-97 3.5 Gly-167 H-Bond Asn-31 2.7 VDW Tyr-32 3.7 Asp-168 H-BondThr-28 2.9 VDW Asn-31 3.3 Phe-27 3.4 Glu-169 H-Bond Asn-31 2.9 Val-170VDW Asn-31 3.5 Ser-319 H-Bond Ser-67 2.7 Gly-68 3.0 VDW Ser-30 3.8Ser-67 3.8 Ile-320 VDW Ser-30 4.0

A model based on the composite structure of two CD73(NDT)/11F11complexes superimposed on CD73 dimer (PDB Entry 4H1 S) suggests that11F11 binds to the surface on CD73 away from the dimer interface,suggesting that the Fab would not interfere with dimer formation.

A comparison of HDX-MS mapping and the X-ray results on the CD73/11F11complex shows that they are in basic agreement showing a similar epitopeon CD73 (65-83 and 157-172). However, the X-ray structure showsadditional interactions (less than 6 Å) in the region of Met-105 toAsp-111 (including H-bonds to Arg-109 and Tyr-110), Lys-135 to Pro-139,and Asp-317 to Ile-320 (including H-bonds to Ser-319).

Example 11: Impact of Different Hinge/Fcs on Size of Antibody/CD73Complexes

As shown in the above Examples, anti-CD73 antibodies with an IgG2 hingeand CH1 are better inhibitors of CD73 enzymatic activity on cells andinternalize better than the same antibodies with an IgG1 hinge. Based onthis observation, and the fact that an IgG2 hinge is stiffer than anIgG1 hinge, it was hypothesized that larger complexes are formed betweenan antigen and antibodies having an IgG2 hinge relative to antibodieshaving an IgG1 hinge. The following experiment was conducted to analyzethis hypothesis.

The structure and oligomeric state of CD73/antibody complexes insolution were examined by SEC-MALS and DLS. For these studies,antibodies containing either an IgG1 or IgG2 constant region, were mixedat varying molar ratios with recombinant proteins comprising either thefull length extracellular domain of human-CD73 containing a C-terminalpolyhistidine tag (amino acid residues 26-546 of human-CD73, termed“hCD73-his”) or a fragment corresponding to the N-terminal domain ofhuman-CD73 (amino acid residues 26-336, termed “N-hCD73-his”).

The oligomeric state of CD73/antibody complexes were examined bysize-exclusion chromatography coupled to an in-line multi-angle lightscattering detector (SEC-MALS). Isocratic separations were performed ona Shodex PROTEIN KW-803 column connected to an Prominence Shimadzu UFLCin buffer containing 200 mM K2HPO₄, 150 mM NaCl, pH 6.8, containing0.02% Na azide (0.1 μm filtered) running at 0.5 mL/min. Samples wereinjected onto the column using a SIL-20AC Prominence Shimadzuautosampler, and data were obtained from three online detectorsconnected in series: a Prominence SPD-20AD diode array UV/visspectrophotometer followed by a Wyatt miniDAWN™ TREOS Multi-Angle LightScattering Detector then a Wyatt Optilab T-rEX Refractive IndexDetector. Data were collected and analyzed using Astra (Wyatt) andLabsolutions (Shimadzu) software.

Dynamic light scattering (DLS) studies were performed on a Wyatt DynaProplate reader in 384 well plates at 25° C. Experimental parameters were20 acquisitions of 5 s each per measurement, and measurements wererecorded in quadruplicate, with the average and standard deviationreported. Intensity autocorrelation functions were fitted using the“Regularization” algorithm in the Dynamics software (WyattTechnologies).

A summary of the SEC-MALS and DLS is provided in FIGS. 29A and B.Analysis of the antibodies alone, shows retention times (about 16-17min), masses (140-150 kDa), and hydrodynamic radii (5.0-5.4 nm) for eachantibody that are typical for a monomeric monoclonal antibody. The datafor the hCD73-his protein is consistent with the protein adopting theexpected dimeric structure in solution; in particular, the massdetermined from the SEC-MALS data (120 kDa) is consistent with thatexpected for a CD73-his dimer (117 kDa) and inconsistent with what wouldbe expected for a hCD73-his monomer (58.5 kDa). The data for N-hCD73 isconsistent with the recombinant N-domain protein being monomeric insolution (SEC-MALS measured mass=38 kDa, compared to expected monomericmass=35.0 kDa), which is expected because the region of the full lengthCD73 extracellular domain that is responsible for dimerization of theprotein is contained within the C-terminal domain without contributionof N-domain residues.

Equimolar mixtures of a given antibody with N-hCD73-his were found toelute as a single species in the SEC with shorter retention time thanthe antibody or N-hCD73-his alone, as well as larger hydrodynamic radii(Rh) by DLS, which is consistent with the formation of complexes. MALSdata indicate masses for these complexes of approximately 210 kDa. Thisis consistent with one N-hCD73-his molecule bound to each of the two Fabdomains of a given antibody to form a 1:2 antibody:N-hCD73-his complex.

SEC-MALS data for mixtures of anti-CD73 antibodies with hCD73-his dimershows that the mixture elutes earlier than either the hCD73-his orantibody alone, suggesting that complexes are formed. Comparing the datafor mAbs that contain the same variable region but different constantdomains, shows that the elution times for the complexes of hCD73-hiswith mAbs containing a IgG2 constant domains (IgG2-C219S,IgG2-C219S-IgG1.1f) are earlier than those for complexes of hCD73-hiswith mAbs containing an IgG1.1f constant domain. In addition, theMALS-determined masses for complexes of hCD73-his with mAbs containingan IgG2 constant domain are larger than those for complexes of hCD73-hiswith mAbs containing an IgG1 constant domain. DLS data further showsthat the hydrodynamic radius of complexes of hCD73-his with mAbscontaining a IgG2 constant domain are larger than those for complexes ofhCD73-his with mAbs containing an IgG1 constant domain. For example, theSEC-MALS and DLS data for CD73.4 with three different constant regions(IgG2-C219S, IgG2-C219S-IgG1.1f, or IgG1.1f) is shown in FIG. 30. Hereit can be seen that the complex of hCD73-his with CD73.4 containing theIgG2 constant domain have shorter retention times (FIG. 30A), largerhydrodynamic radii (FIG. 30B) and larger MALS-determined masses (FIG.30C), as compared to the complexes of hCD73-his with CD73.4-IgG1.1f.Based on the MALS masses, a schematic model for the structure andstoichiometry of the complexes between hCD73-his and the antibodies isshown in FIG. 30D, where complexes containing CD73.4-IgG1.1fpredominantly form smaller 2:2 (peak 1=˜550 kDa) or 4:4 mAb/CD73 dimercomplexes (peak 2=˜1300 kDa), whereas CD73.4-IgG2-C219S orCD73.4-IgG2-C219S-IgG1.1f form much larger complexes (>3000 kDa) withhCD73-his, for which precise structure and stoichiometry cannot beconfidently modeled.

CD73.4 antibodies having the heavy chain constant regions set forth inTable 26 were also tested for size of complex formation. As shown inFIG. 30D, the results indicate that higher order complexes are formedwith antibodies having an IgG2 CH1 domain relative to those having anIgG1 CH1 domain.

Collectively the SEC-MALS and DLS data demonstrate that larger complexesare formed between hCD73-his and mAbs containing an IgG2 hinge and CH1region (IgG2-C219S or IgG2-C219S-IgG1.1f), compared to those containingthe IgG1 hinge and CH1 region (IgG1.1f). In addition, antibodies havingan IgG2 CH1 domain form larger complexes that those having an IgG1 CH1domain.

Example 12: Relevance of Certain Amino Acid Residues in IgG2 CH1 andHinge in Improving Antibody Mediated CD73 Internalization

Anti-CD73 antibodies (CD73.4) with the heavy chain constant regionsshown in Table 31 were prepared and tested as described above inantibody mediated CD73 internalization assays.

TABLE 31Heavy chain constant regions that were fused to anti-CD73 variable regionsSEQ ID NO Description Constructs of constant regionCH1 domain of IgG2, with all else IgG1. G2-G1-G1-G1 300 Also, Cys >Ser mutant to reduce potential disulfide G2.5-G1-G1-G1 301heterogeneity: CH1 domain of IgG1 with all else IgG2.3: G1-G2.3-G2-G2302 Swap CH1 regions in IgG1 with those of IgG2, either G1-KRGEGSSNLF303 separate or together G1-KRGEGS 304 G1-SNLF 305 IgG1-ITNDRTPR 306G1-SNLFPR 307 Swap CH1 regions in IgG2 with those of IgG1, eitherG2-RKEGSGNSFL 308 separate or together: G2-RKEGSG 309 G2-NSFL 310IgG2-TIDNTRRP 311 G2-NSFLRP 312 IgG1 with CH2 domain residues of IgG2:G1-G1-G2-G1-AY 313 G1-G1-G2-G1-KH 314IgG2 with CH2 domain residues of IgG1: G2-G2.3-G1-G2-KH 315G2.5-G2.3-G1-G2-KH 316 G2-G2.3-G1-G2-AY 317 G2.5-G2.3-G1-G2-AY 318Swap hinge regions between IgG1 and IgG2: G1-G2.3-G1-G1-KH 319G2-G1-G2-G2-AY 320 G2.5-G1-G2-G2-AY 321 G1-G2-G1-G1-AY 322G2-G1-G2-G2-KH 323 G2.5-G1-G2-G2-KH 324 Hinge truncationsIgG1-deltaHinge 325 IgG2-deltaHinge 326 IgG2.5-deltaHinge 327IgG1-deltaG237 328 IgG2-plusG237 329 Other IgG2.4 330 IgG2.3/4 331The results, which are shown in FIG. 31, provide the followinginformation in the context of CD73 internalization:

-   -   CH2 domain does not appear to have an impact as shown by        -   a) very little difference in internalization ability was            observed between the antibodies comprising a modified heavy            chain constant region with format “AY” (having the IgG2            hinge ERKCCVECPPCPAPPVAG (SEQ ID NO: 8) relative to those            with format “KH” (ERKCCVECPPCPAPEL (SEQ ID NO: 22) (Set 5, 6            and 7);        -   b) CH2 swaps are comparable to wildtype G1 or G2 (Sets 5 and            6); and        -   c) residue 237 has no impact on internalization: neither the            addition of a “G” residue to an IgG2 hinge nor the deletion            of the C terminal “G” in an IgG hinge affected            internalization (Set 9).    -    This suggests that the CH2 domain does not impact        internalization (i.e., the CH2 domain can be from IgG1 or IgG2);    -   the CH1 regions indicated in Set 3 (KRGEGSSNLF; KRGEGS; SNLF;        ITNDRTPR and SNLFPR) in IgG1 with those of IgG2 provides little        benefit, i.e., the internalization remains similar to that of        IgG1; see Set 3);    -   Swapping the CH1 regions indicated in Set 4 (RKEGSGNSFL; RKEGSG;        NSFL; TIDNTRRP and NSFLRP) in IgG2 with those of IgG1 has        variable impact: changing NSFL has no impact, whereas the other        2 regions (RKEGSG & RP) are involved (see Set 4). Based on the        results of Sets 3 and 4, it appears that there is an interaction        between the CH1 region and the hinge, with RKEGSG and RP regions        being more important than NSFL region;    -   The hinge region impacts internalization, i.e., the hinge of        IgG2 provides better internalization relative to the hinge of        IgG (see Sets 7 and 8). In addition, IgG with a deletion        (G1-delta-hinge) improves internalization over IgG1. IgG2 with a        deletion (G2-delta-hinge) provides a similar level of        internalization relative to that of an IgG2 hinge. This suggests        that the hinge region impacts internalization, which effect is        enhanced by an IgG2 CH1 (G2-G1-G2-G2-AY is comparable to        G1-G2-G1-G1-AY);    -   IgG2.4 (C220S) has similar or reduced internalization compared        to IgG2.3 (C219S). IgG2.3/4 (C219S/C220S) has much reduced        internalization compared to IgG2.3 or IgG2.4 alone (see Set 10).        This suggests that internalization of an antibody with an IgG2        hinge and C219S is about the same as that of an IgG2 hinge with        C220S, both of which are much better than that of an IgG2 hinge        with both C219S and C220S;    -   IgG2.5 (C131S mutation) has reduced internalization compared to        constructs with C131 (see Sets 1, 6 and 7).

Thus, these results indicate that the CH1 domain and the hinge are bothrelevant in the antibody mediated CD73 internalization, and that anantibody having the IgG2 sequences from these domains is internalizedwith better efficacy relative to an antibody having these regions fromIgG1.

Example 13: Antibodies Having an IgG2 Hinge and/or CH1 Domain Form HighMolecular Weight Complexes

CD73.4 antibodies having the heavy chain constant regions set forth inTable 26 were also tested for formation of high molecular weightcomplexes by SEC-MALS and DLS experiments, as described in Example 11.

Three out of the 16 antibodies in this study were were previouslytested: CD73.4-IgG1.1f, CD73.4-IgG2-C219S (also called CD73.4-IgG2.3),and CD73.4-IgG2-C219S-IgG1.1f (also called CD73.4-IgG2.3G1.1f-KH).SEC-MALS and DLS data of the antibodies alone showed retention times,masses, and hydrodynamic radii for each antibody that are typical for amonomeric monoclonal antibody. Equimolar complexes of each antibody (5.5uM) with hCD73-his (5.5 uM) showed slower retention times for allcomplexes as compared to antibody or hCD73-his alone indicating theformation of complexes. An overlay of the SEC chromatogram data for eachof the 16 complexes is shown in FIG. 32A. The chromatogram data can bedivided into 4 distinct peaks, which are shown in FIG. 32B. Peak 1contains the largest species, with MALS-determined masses suggestingcomplexes with mass equivalent of greater than 4:4 hCD73-his:mAbcomplexes. Peak 2 contains species with MALS-determined massessuggesting complexes of about 2:2 hCD73-his:mAb complexes. Peak 3 is aminor species with low signal and MALS-determined masses suggestingabout 1:1 hCD73-his:mAb complexes. Peak 4 corresponds to the elution ofthe mAbs alone with MALS-determined masses consistent with freeantibody. To quantitate the relative amounts of each species, the 4peaks of each chromatogram were integrated as peak 1 (<12.9 min), peak 2(12.9-15.1 min), peak 3 (15.1-16.7 min), peak 4 (16.7-19.3 min). Theintegration also included an additional integrated range called peak 5(>19.3 min) to account for any low molecular weight species, which werefound to be negligible (<3.5% for all complexes). The percentage of eachspecies from this integration is summarized in Table 32. All complexescontained a similar small percentage of peak 3 (about 6-9%), butvariable amounts of the other peaks. Most notable is that all complexesbetween hCD73-his and antibodies containing a CH1 domain from hIgG1 hada significantly greater percentage of smaller complexes (peak 2),whereas those containing CH1 domain from hIgG2 had a greater percentageof larger complexes (peak 1) (Table 32 and FIG. 32C). This suggests animportant role for not only the hinge region but also the CH1 domain inhigher order complex formation.

TABLE 32 Retention times of CD73.4 antibodies with modified heavy chainconstant regions UV % Peak2 Peak3 Peak4 Peak1 12.9- 15.1- 16.7- Peak5Complexes <12.9 min 15.1 min 16.7 min 19.3 min >19.3 min CD73.4-IgG2.3 +hCD73-his 37.0 23.8 7.7 28.6 2.9 CD73.4-IgG2.3G1.1f-KH + hCD73-his 36.023.8 7.9 29.3 3.0 CD73.4-IgG1.1f + hCD73-his 28.4 36.2 7.4 25.6 2.3CD73.4-IgG1f + hCD73-his 26.0 36.5 7.5 27.8 2.2 CD73.4-IgG2.3G1-AY +hCD73-his 30.2 24.3 8.1 34.4 3.0 CD73.4-IgG2.3G1-KH + hCD73-his 34.923.4 7.9 30.7 3.0 CD73.4-IgG1-G2.3G1-AY + hCD73-his 14.6 29.2 6.4 48.31.6 CD73.4-IgG1-G2.3G1-KH + hCD73-his 23.8 32.6 7.0 34.5 2.1CD73.4-IgG1-deltaTHT + hCD73-his 28.3 35.4 7.0 26.9 2.4CD73.4-IgG2.3-plusTHT + hCD73-his 30.6 24.3 8.3 33.7 3.2CD73.4-IgG2.3-plusGGG + hCD73-his 30.0 23.9 8.2 34.9 2.9 CD73.4-IgG2.5 +hCD73-his 31.7 24.4 8.4 32.5 3.1 CD73.4-IgG2.5G1.1f-KH + hCD73-his 30.724.3 8.9 32.7 3.4 CD73.4-IgG2.5G1-AY + hCD73-his 26.3 24.8 8.1 38.3 2.6CD73.4-IgG2.5G1-KH + hCD73-his 21.4 24.1 7.0 45.6 1.9CD73.4-IgG2.5-plusTHT + hCD73-his 32.6 23.5 8.3 32.6 3.0

Example 14: Fc Receptor Binding for Antibodies with Engineered ConstantDomains

This Example demonstrates that antibodies having modified heavy chainconstant regions comprising the CH1 and hinge of IgG2 bind to FcγRs whenthey contain CH2 and CH3 domains of IgG1.

In addition to antigen binding by the variable domains, antibodies canengage Fc-gamma receptors (FcgRs) through interaction with the constantdomains. These interactions mediate effector functions such asantibody-dependent cellular cytotoxicity (ADCC) and antibody-dependentcellular phagocytosis (ADCP). Effector function activity is high for theIgG1 isotype, but very low or absent for IgG2 and IgG4 due to theseisotypes having lower affinity for FcgRs. In addition, the effectorfunction of IgG1 can be modified through mutation of amino acid residueswithin the constant regions to alter FcgR affinity and selectivity.

The binding of antibodies to Fc gamma receptors (FcγRs or FcgRs) wasstudied using biosensor technologies including Biacore surface plasmonresonance (SPR) and Fortebio Biolayer Interferometry (BLI). SPR studieswere performed on a Biacore T100 instrument (GE Healthcare) at 25° C.The Fab fragment from a murine anti-6×His antibody was immobilized on aCM5 sensor chip using EDC/NHS to a density of ˜3000 RU. Varioushis-tagged FcgRs (7 ug/ml) were captured via the C-terminal his-tagusing a contact time of 30 s at 10 ul/min, and the binding of 1.0 μMantibody was evaluated in a running buffer of 10 mM NaPO4, 130 mM NaCl,0.05% p20 (PBS-T) pH 7.1. FcgRs used for these experiments included CD64(FcgRI), CD32a-H131 (FcgRIIa-H131), CD32a-R131 (FcgRIIa-R131), CD32b(FcgRIIb), CD16a-V158 (FcgRIIIa-V158), CD16b-NA1 (FcgRIIIb-NA1), andCD16B-NA2 (FcgRIIIb-NA2). BLI experiments were performed on a FortebioOctet RED instrument (Pall, Fortebio) at 25° C. in 10 mM NaPO4, 130 mMNaCl, 0.05% p20 (PBS-T) pH 7.1. Antibodies were captured out ofundiluted expression supernatants on protein A coated sensors, followedby the binding of 1 μM hCD32a-H131, hCD32a-R131, hCD32b, hCD16a-V158, or0.1 μM hCD64 analytes.

First, antibodies were made that contain modified IgG1 Fc domainsincluding the substitutions S267E (SE) and S267E/L328F (SELF), as wellas various combinations of the mutations P238D, P271G, H268D, A330R,G237D, E233D, referred to as V4, V7, V8, V9 and V12. The binding ofthese antibodies was studied by Biacore SPR with comparison to IgG1f,IgG2.3 (IgG2-C219S) and IgG4.1 (IgG4-S228P) antibodies, as well as anIgG1.1f antibody which has been engineered to reduce binding to allFcgRs. The results, which are shown in FIG. 33, demonstrate the expectedFcgR binding properties for IgG1f, IgG2.3 and IgG4.1 and the mutatedIgG1 antibodies, including increased CD32a-H131, CD32a-R131 and CD32bbinding for SE and SELF, as well as increased selectivity of the V4, V7,V8, V9 and V12 mutants for CD32b over CD32a-H131 and CD32a-R131, FIG.33.

The next set of constructs was used to engineer effector function intothe otherwise effector function negative IgG2 isotype. For this study,the mutations described above were introduced in the context of anIgG2.3 constant region, or an IgG2.3/IgG1f hybrid termed IgG2.3G1-AY(Table 33). Antibodies were expressed at small scale as supernatants,and tested for binding to FcgRs using Fortebio Octet BioLayerInterferometry biosensor technology. Since the antibodies were presentat low concentration in the supernatants, the experiment was performedby capturing antibodies out of the supernatants using protein A coatedsensors, followed by binding of FcgR analytes in solution. Purified andsupernatant control IgG1f including wild type IgG1, SE, P238D, V4 andV12 antibodies were also included for comparison, and each of thesecontrol antibodies demonstrated expected FcgR binding properties (FIG.34). The IgG2.3 antibody also demonstrated the expected binding profile,with appreciable binding to only CD32a-H131. However, all mutations tointroduce S267E, L328F, P238D, P271G, H268D, A330R, G237D, or E233Dmutations into IgG2.3 failed to recapitulate the FcgR affinity of thecorresponding engineered IgG1 mAbs (FIG. 34). In contrast, theIgG2.3G1-AY construct was able to fully preserve the FcgR bindingproperties of wild type IgG1, while retaining the CH1 and hinge regionsof IgG2.3. In addition, all IgG2.3G1-AY mutants containing S267E, L328F,P238D, P271G, H268D, A330R, G237D, and E233D demonstrated FcgR bindingproperties comparable to the IgG1 version mAbs containing the samemutations (FIG. 34). This demonstrates the successful engineering ofantibodies with CH1 and hinge regions of IgG2 combined with the effectorfunction of wild type or mutant IgG1.

TABLE 33 Engineered IgG2 constructs Set ID Construct Seq ID# 1 IgG2.3hHC-IgG2-C219S 268 IgG2.3-V13 hHC-IgG2-C219S - P238D 332 IgG2.3-V14hHC-IgG2-C219S - P238D, 333 P271G IgG2.3-V15 hHC-IgG2-C219S - P238D, 334H268D, P271G IgG2.3-V16 hHC-IgG2-C219S - P238D, 335 P271G, A330RIgG2.3-V17 hHC-IgG2-C219S - P238D, 336 H268D, P271G, A330R IgG2.3-V18hHC-IgG2-C219S - S267E 337 IgG2.3-V19 hHC-IgG2-C219S - S267E, L328F 3382 IgG2.3G1 hHC-IgG2-C219S/hHC-IgG1f 269 IgG2.3G1-AY-V20hHC-IgG2-C219S/hHC-IgG1f - 339 P238D IgG2.3G1-AY-V21hHC-IgG2-C219S/hHC-IgG1f - 340 P238D, P271G IgG2.3G1-AY-V22hHC-IgG2-C219S/hHC-IgG1f - 341 P238D, H268D, P271G IgG2.3G1-AY-V23hHC-IgG2-C219S/hHC-IgG1f - 342 P238D, P271G, A330R IgG2.3G1-AY-V24hHC-IgG2-C219S/hHC-IgG1f - 343 P238D, H268D, P271G, A330RIgG2.3G1-AY-V25 hHC-IgG2-C219S/hHC-IgG1f - 344 G237D, P238D, H268D,P271G, A330R IgG2.3G1-AY-V26 hHC-IgG2-C219S/hHC-IgG1f - 345 E233D,G237D, P238D, H268D, P271G, A330R IgG2.3G1-AY-V27hHC-IgG2-C219S/hHC-IgG1f - 346 S267E IgG2.3G1-AY-V28hHC-IgG2-C219S/hHC-IgG1f - 347 S267E, L328F

This engineering strategy was further explored by producing otherantibodies formatted with IgG2.3G1-AY, IgG2.3G1-AY-S267E(IgG2.3G1-AY-V27), as well as IgG2-B-form variants (IgG2.5G1-AY andIgG2.5G1-AY-V27), and other hybrid antibodies containing differentcombinations of IgG1 and IgG2 constant domains, and testing the bindingof these antibodies to anti-his Fab captured his-tagged FcgRs usingBiacore SPR technology. In agreement with the Octet supernatant data,the SPR data showed that the IgG2.3G1-AY and IgG2.3G1-AY-V27 antibodieshad comparable FcgR binding properties to IgG1f and IgG1f-S267E,respectively, despite containing the CH1 and hinge regions of an A-formIgG2 antibody (IgG2.3) (Table 34). Similar data was also obtained usingIgG2.5G1-AY and IgG2.5G1-AY-V27 antibodies, demonstrating the successfulengineering of B-form IgG2 antibodies (containing C131S mutation termedIgG2.5) having IgG1f or modified IgG1f like effector functions. Data forseveral other antibodies with IgG2.3G1-AY, IgG2.3G1-AY-V27, IgG2.5G1-AY,or IgG2.5G1-AY-V27 constant regions but different variable regionsshowed that this engineering strategy is broadly applicable to otherantibodies independent of the variable domains (Table 34). Otherconstructs that demonstrate IgG1f-like FcgR binding properties includeIgG1-G2.3G1-AY, and IgG1deltaTHT, whereas several of the modifiedconstant region constructs were unable to retain IgG1f-like FcgR bindingproperties, including IgG2.3G1-KH, IgG2.5G1-KH, IgG2.3plusTHT,IgG2.5plusTHT and IgG2.3plusGGG constructs (Table 34).

TABLE 34 % Rmax values for 1 μM antibody binding to anti-his Fabcaptured FcgR-his proteins hCD32a- hCD32a- hCD16a- hCD16B- mAb hCD64H131 R131 hCD32b V158 NA2 mAb8-IgG1f 80%  82% 51% 27% 51% 21% mAb9-IgG1f70%  33% 19%   4% 28% 10% CD73.4-IgG1f 65%  46% 26%   6% 43% 17%CD73.4-IgG1.1f  2%  0%  2%   1%  0%   0% mAb11-IgG2.3  2%  44% 17%   5% 1%   0% CD73.4-IgG2.3  3%  48% 11%   1%  1%   0% mAb6-IgG2.3  3%  66%14%   3%  1%   0% mAb4-IgG2.3  1%  39%  6%   1%  1%   0% mAb5-IgG2.3  6%100% 30%   4%  3%   0% mAb12-IgG2.3  2%  39%  7%   1%  1%   0%mAb13-IgG2.3  2%  40%  7%   1%  1%   0% mAb11-IgG2.5  0%  40% 13%   3% 0% −1% mAb7-IgG2.5  4%  72% 19%   2%  2%   0% mAb8-IgG2.5  3%  59% 14%  3%  2%   0% mAb10-IgG2.5  1%  29%  5%   1%  1%   0% CD73.4-IgG2.5  3% 40%  7%   1%  1%   0% mAb6-IgG2.5  3%  75% 17%   4%  2%   0%mAb4-IgG2.5  2%  46%  8%   1%  1%   0% mAb5-IgG2.5  6%  89% 26%   5%  4%  1% mAb12-IgG2.5  1%  36%  6%   1%  1%   0% mAb13-IgG2.5 −2%  39%  4%−2%  0% −2% mAb8-IgG2.3G1-AY 77%  61% 38% 10% 38% 13% mAb10-IgG2.3G1-AY67%  23% 14%   4% 24%   8% CD73.4-IgG2.3G1-AY 65%  38% 20%   5% 38% 14%mAb7-IgG2.5G1-AY 80%  73% 45% 12% 47% 19% mAb8-IgG2.5G1-AY 77%  70% 45%17% 48% 22% CD73.4-IgG2.5G1-AY 65%  43% 24%   7% 40% 16%CD73.4-IgG2.3G1-KH  2%  15%  2%   0%  2%   0% CD73.4-IgG2.5G1-KH  2% 17%  2%   0%  3%   0% CD73.4-IgG2.3G1.1f-KH  1%  10%  1%   0%  1%   0%CD73.4-IgG2.5G1.1f-KH  1%  6%  1%   0%  1%   0% mAb7-IgG2.3G1-AY-V27 84% 68% 92% 76% 26%   7% mAb8-IgG2.3G1-AY-V27 78%  67% 80% 67% 24%   7%mAb10-IgG2.3G1-AY-V27 69%  24% 57% 40% 12%   3% mAb7-IgG2.5G1-AY-V27 81% 74% 89% 84% 32%   9% mAb8-IgG2.5G1-AY-V27 77%  76% 79% 77% 33% 10%CD73.4-IgG1-G2.3G1-AY 66%  50% 31% 10% 48% 23% CD73.4-IgG1-G2.3G1-KH  2% 18%  2%  0%  4%   1% CD73.4-IgG1deltaTHT 65%  43% 23%  6% 42% 17%CD73.4-IgG2.3plusTHT  3%  42%  8%  1%  1%   0% CD73.4-IgG2.5plusTHT  2% 34%  7%  1%  1%   0% CD73.4-IgG2.3plusGGG  3%  43%  8%  1%  1%   0%Taken together these data show that the sequence in IgG1 immediatelyC-terminal to the conserved CPPCPAP (SEQ ID NO: 380) motif in the hingeregion confers FcgR-mediated effector function, whereas the CH1 andupper portions of the hinge of the antibody can be replaced with IgG2 ormodified IgG2 sequences, to potentially combine the effector functionsof IgG1 and modified IgG1 with the superior internalization or signalingproperties of antibodies containing IgG2 CH1 and/or hinge regions.

EQUIVALENTS

Those skilled in the art will recognize or be able to ascertain, usingno more than routine experimentation, many equivalents of the specificembodiments described herein described herein. Such equivalents areintended to be encompassed by the following claims.

SUMMARY OF SEQUENCE LISTING

TABLE 35 SEQ ID Description Sequence   1 Human CD73 MCPRAARAPA TLLLALGAVL WPAAGAWELT isoform 1ILHTNDVHSR LEQTSEDSSK CVNASRCMGG VARLFTKVQQ IRRAEPNVLL LDAGDQYQGTIWFTVYKGAE VAHFMNALRY DAMALGNHEFDNGVEGLIEP LLKEAKFPILSANIKAKGPL ASQISGLYLP YKVLPVGDEV VGIVGYTSKE TPFLSNPGTN LVFEDEITALQPEVDKLKTL NVNKIIALGH SGFEMDKLIA QKVRGVDVVV GGHSNTFLYT GNPPSKEVPAGKYPFIVTSD DGRKVPVVQA YAFGKYLGYL KIEFDERGNV ISSHGNPILL NSSIPEDPSIKADINKWRIK LDNYSTQELG KTIVYLDGSS QSCRFRECNM GNLICDAMIN NNLRHTDEMFWNHVSMCILN GGGIRSPIDE RNNGTITWEN LAAVLPFGGT FDLVQLKGST LKKAFEHSVHRYGQSTGEFL QVGGIHVVYD LSRKPGDRVV KLDVLCTKCRVPSYDPLKMD EVYKVILPNFLANGGDGFQM IKDELLRHDS GDQDINVVST YISKMKVIYP AVEGRIKFST GSHCHGSFSLIFLSLWAVIF VLYQ   2 Human CD73  MCPRAARAPA TLLLALGAVL WPAAGAWELTisoform 2 ILHTNDVHSR LEQTSEDSSK CVNASRCMGGVARLFTKVQQ IRRAEPNVLLLDAGDQYQGT IWFTVYKGAE VAHFMNALRY DAMALGNHEFDNGVEGLIEP LLKEAKFPILSANIKAKGPL ASQISGLYLP YKVLPVGDEV VGIVGYTSKETPFLSNPGTN LVFEDEITALQPEVDKLKTL NVNKIIALGH SGFEMDKLIA QKVRGVDVVVGGHSNTFLYT GNPPSKEVPAGKYPFIVTSD DGRKVPVVQA YAFGKYLGYL KIEFDERGNVISSHGNPILL NSSIPEDPSIKADINKWRIK LDNYSTQELG KTIVYLDGSS QSCRFRECNMGNLICDAMIN NNLRHTDEMFWNHVSMCILN GGGIRSPIDE RNNGIHVVYD LSRKPGDRVVKLDVLCTKCR VPSYDPLKMDEVYKVILPNF LANGGDGFQM IKDELLRHDS GDQDINVVSTYISKMKVIYP AVEGRIKFSTGSHCHGSFSL IFLSLWAVIF VLYQ   3 Cynomolgus CD73MCPRAARAPA TLLLAVGALL WSAAGAWELT ILHTNDVHSR LEQTSEDSSKCVNASRCMGGVARLFTKVQQ IRRAEPNVLL LDAGDQYQGT IWFTVYKGAE VAHFMNALRYDAMALGNHEFDNGVEGLIEP LLKEAKFPIL SANIKAKGPL ASQISGLYLP YKVLPVGDEVVGIVGYTSKETPFLSNPGTN LVFEDEITAL QPEVDKLKTL NVNKIIALGH SGFETDKLIAQKVRGVDVVVGGHSNTFLYT GNPPSKEVPA GKYPFIVTSD DGRKVPVVQA YAFGKYLGYLKIEFDERGNVISSHGNPILL NSSIPEDPSI KADINKWRIK LDNYSTQELG KTIVYLDGSSQSCRFRECNMGNLICDAMIN NNLRHADEMF WNHVSMCILN GGGIRSPIDE RNNGTITWENLAAVLPFGGTFDLVQLKGST LKKAFEHSVH RYGQSTGEFL QVGGIHVVYD LSRKPGDRVVKLDVLCTKCRVPSYDPLKMD EIYKVILPNF LANGGDGFQM IKDELLRHDS GDQDINVVSTYISKMKVIYPAVEGRIKFST GSHCHGSFSL IFLSFCAVIF VLYQ   4 11F11 VHQVQLVESGGGVVQPGRSLRLSCATSGFTFSNYGMH WVRQAPGKGLEWVAVILYDGSNKYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSSW YPDSFDIWGQGTMVTVSS   5 11F11 VH CDR1NYGMH   6 11F11 VH CDR2 VILYDGSNKYYPDSVKG   7 11F11 VH CDR3 GGSSWYPDSFDI  8 11F11 VK1 EIVLTQSPATLSLSPGERATLSCRASQGVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGPGTDFT LTISSLEPEDFAVYYCQQRSNWHLTFGGGTKVEIK  9 11F11 VK1 CDR1 RASQGVSSYLA  10 11F11 VK1 CDR2 DASNRAT  1111F11 VK1 CDR3 QQRSNWHLT  12 11F11 VK2DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQYNSYPLTFGGGTKVEIK 13 11F11 VK2 CDR1 RASQGISSWLA  14 11F11 VK2 CDR2 AASSLQS  1511F11 VK2 CDR3 QQYNSYPLT  16 4C3 VH EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWKSGSIGYADSVKGRF TISRDNAKNSLYLQMNSLRAEDTALYYCVKGYYVILTGLDYWGQGTLVTVSS  17 4C3 VH CDR1 DYAMH  18 4C3 VH CDR2GISWKSGSIGYADSVKG  19 4C3 VH CDR3 GYYVILTGLDY  20 4C3 VK1EIVLTQSPGTLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFT LTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIK 21 4C3 VK1 CDR1 RASQSVSSYLAW  22 4C3 VK1 CDR2 ASSRATG  23 4C3 VK1 CDR3QYGSSPLT  24 4C3 VK2 DIQMTQSPSSLSASVGDRVTFTCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPPTFGQGTKVEIK  25 4C3 VK2 CDR1 RASQGISSWLA  264C3 VK2 CDR2 AASSLQS  27 4C3 VK2 CDR3 QQYNSYPPT  28 4C3 VK3DIQMTQSPSSLSASVGDRVTFTCRASQGISSWLAW YQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPPTFGQGTKVEIK  29 4C3 VK3 CDR1 RASQGISSWLA  304C3 VK3 CDR2 AASSLQS  31 4C3 VK3 CDR3 QQYNSYPPT  32 4D4 VHQVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGM HWVRQAPGKGLEWVAVIWYDESNKYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCARGYNS RWYPDAFDIWGQGTMVT VSS  334D4 VH CDR1 NYGMH  34 4D4 VH CDR2 VIWYDESNKYYADSVKG  35 4D4 VH CDR3GYNSRWYPDAFDI  36 4D4 VK1 DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQYNSYPLTFGGGTKVEIK 37 4D4 VK1 CDR1 RASQGISSWLA  38 4D4 VK1 CDR2 AASSLQS  39 4D4 VK1 CDR3QQYNSYPLT  40 10D2 VH1 QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGLHWVRQAPGKGLEWVAVIRYDGSNKYYADSVKGRF TISRDNSKNTLYLQMSSLRAEDTAVYYCARGGSSWYPDGLDVWGQGTTVTV SS  41 10D2 VH1 CDR1 NYGLH  42 10D2 VH1 CDR2VIRYDGSNKYYADSVKG  43 10D2 VH1 CDR3 GGSSWYPDGLDV  44 10D2 VK1AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQFNSYPTFGGGTKVEIK 45 10D2 VK1 CDR1 RASQGISSALA  46 10D2 VK1 CDR2 DASSLES  4710D2 VK1 CDR3 QQFNSYPT  48 10D2 VK2 DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQYNSYPLTFGGGTKVEIK 49 10D2 VK2 CDR1 RASQGISSWLA  50 10D2 VK2 CDR2 AASSLQS  5110D2 VK2 CDR3 QQYNSYPLT  52 11A6 VH EVQLVESGGNLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNNNDIGYADSVKGRF IISRDNAKNSLYLQMNSLRPEDTALYYCVKGYYVILTGLDYWGQGTPVTVS S  53 11A6 VH CDR1 DYAMH  54 11A6 VH CDR2GISWNNNDIGYADSVKG  55 11A6 VH CDR3 GYYVILTGLDY  56 11A6 VK1DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQYNSYPLTFGGGTKVEIK 57 11A6 VK1 CDR1 RASQGISSWLA  58 11A6 VK1 CDR2 AASSLQS  5911A6 VK1 CDR3 QQYNSYPLT  60 24H2 VH QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVIWYDGGNKYYADSVKG RFTISRDNSKNTLFLQMNSLRAEDTAVYYCARGGSSWYPDAFDIWGQGTMVTV SS  61 24H2 VH CDR1 NYGMH  62 24H2 VH CDR2VIWYDGGNKYYADSVKG  63 24H2 VH CDR3 GGSSWYPDAFDI  64 24H2 VK1DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQYNSYPLTFGGGTKVEIK 65 24H2 VK1 CDR1 RASQGISSWLA  66 24H2 VK1 CDR2 AASSLQS  6724H2 VK1 CDR3 QQYNSYPLT  68 5F8 VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMHWVRQAPGKGLVWVSRIISDGSSTGYADSVKGRFTI SRDNAKNTLYLQMNSLRAEDTAVYYCAREFSSGWYFDYWGQGTLVTVSS  69 5F8 VH CDR1 SYWMH  70 5F8 VH CDR2 RIISDGSSTGYADSVKG 71 5F8 VH CDR3 EFSSGWYFDY  72 5F8 VK1AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQFSSYPRTFGQGTKVEIK 73 5F8 VK1 CDR1 RASQGISSALA  74 5F8 VK1 CDR2 DASSLES  75 5F8 VK1 CDR3QQFSSYPRT  76 5F8 VK2 DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTGFT LTISSLQPEDFATYYCQQYNSYPRTFGQGTKVEIK 77 5F8 VK2 CDR1 RASQGISSWLA  78 5F8 VK2 CDR2 AASSLQS  79 5F8 VK2 CDR3QQYNSYPRT  80 6E11 VH EVQLVESGGALVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGITWNSGGIGYADSVKGRF TISRDNAKNSLYLQMNSLRAEDTALYYCAKDRYYSSWLLFDNWGQGILVTV SS  81 6E11 VH CDR1 DYAMH  82 6E11 VH CDR2GITWNSGGIGYADSVKG  83 6E11 VH CDR3 DRYYSSWLLFDN  84 6E11 VK1EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDF TLTISRLEPEDFAVYYCQHYGSSFTFGPGTKVDIK 85 6E11 VK1 CDR1 RASQSVSSSYLA  86 6E11 VK1 CDR2 GASSRAT  876E11 VK1 CDR3 QHYGSSFT  88 7A11 VH EVQLVESGGGLVQTGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSDISWNSDIIGYADSVKGRF TISRDNAKNSLYLQMNSLRAEDTALYYCAKDIYGSGSSFFDYWGQGILVTV SS  89 7A11 VH CDR1 DYAMH  90 7A11 VH CDR2DISWNSDIIGYADSVKG  91 7A11 VH CDR3 DIYGSGSSFFDY  92 7A11 VK1DIQMTQSPSSLSASVGDRVTITCRASQYISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQYHSYPPTFGQGTRLEIK 93 7A11 VK1 CDR1 RASQYISSWLA  94 7A11 VK1 CDR2 AASSLQS  957A11 VK1 CDR3 QQYHSYPPT  96 11F11 epitope #1 FTKVQQIRRAEPNVLLLDA  9711F11 epitope #2 LYLPYKVLPVGDEVVG  98 Wildtype IgG1 CH1ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV  99 His-tagged CD73MCPRAARAPATLLLALGAVLWPAAGAWELTILHT NDVHSRLEQTSEDSSKCVNASRCMGGVARLFTKVQQIRRAEPNVLLLDAGDQYQGTIWFTVYKGAEVA HFMNALRYDAMALGNHEFDNGVEGLIEPLLKEAKFPILSANIKAKGPLASQISGLYLPYKVLPVGDEVVGIVGYTSKETPFLSNPGTNLVFEDEITALQPEVDKLKT LNVNKIIALGHSGFEMDKLIAQKVRGVDVVVGGHSNTFLYTGNPPSKEVPAGKYPFIVTSDDGRKVPVVQAYAFGKYLGYLKIEFDERGNVISSHGNPILLNSSIPEDPSIKADINKWRIKLDNYSTQELGKTIVYLDGSSQS CRFRECNMGNLICDAMINNNLRHADETFWNHVSMCILNGGGIRSPIDERNNGTITWENLAAVLPFGGTFD LVQLKGSTLKKAFEHSVHRYGQSTGEFLQVGGIHVVYDLSRKPGDRVVKLDVLCTKCRVPSYDPLKMDE VYKVILPNFLANGGDGFQMIKDELLRHDSGDQDINVVSTYISKMKVIYPAVEGRIKHHHHHH 100 11F11 (full length QVQLVESGGGVVQPGRSLRLSCATSGFTFSNYGMH heavy chain)WVRQAPGKGLEWVAVILYDGSNKYYPDSVKGRFT ISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSSWYPDSFDIWGQGTMVTVSSASTKGPSVFPLAPCSRST SESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHK PSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYV DGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK 10111F11 (full length  EIVLTQSPATLSLSPGERATLSCRASQGVSSYLAWY light chain 1)QQKPGQAPRLLIYDASNRATGIPARFSGSGPGTDFTLTISSLEPEDFAVYYCQQRSNWHLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 102 11F11 (full lengthDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWY light chain 2)QQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 103 4C3 (full length EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAM heavy chain)HWVRQAPGKGLEWVSGISWKSGSIGYADSVKGRF TISRDNAKNSLYLQMNSLRAEDTALYYCVKGYYVILTGLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 104 4C3 (full length EIVLTQSPGTLSLSPGERATLSCRASQSVSSYLAWY light chain 1)QQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 105 4C3 (full length DIQMTQSPSSLSASVGDRVTFTCRASQGISSWLAW light chain 2)YQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC 106 4C3 (full length DIQMTQSPSSLSASVGDRVTFTCRASQGISSWLAW light chain 3)YQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC 107 4D4 (full length QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGM heavy chain)HWVRQAPGKGLEWVAVIWYDESNKYYADSVKGR FTISRDNSKNTLFLQMNSLRAEDTAVYYCARGYNSRWYPDAFDIWGQGTMVTVSSASTKGPSVFPLAPCS RSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVD HKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNW YVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 1084D4 (full length  DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWY light chain 1)QQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 109 10D2 (full length QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGLH heavy chain)WVRQAPGKGLEWVAVIRYDGSNKYYADSVKGRF TISRDNSKNTLYLQMSSLRAEDTAVYYCARGGSSWYPDGLDVWGQGTTVTVSSASTKGPSVFPLAPCSRS TSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 110 10D2 (full length AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWY light chain 1)QQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 111 10D2 (full length DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWY light chain 2)QQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 112 11A6 (full length EVQLVESGGNLVQPGRSLRLSCAASGFTFDDYAM heavy chain)HWVRQAPGKGLEWVSGISWNNNDIGYADSVKGRF IISRDNAKNSLYLQMNSLRPEDTALYYCVKGYYVILTGLDYWGQGTPVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 113 11A6 (full length DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWY light chain 1)QQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 114 24H2 (full length QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGM heavy chain)HWVRQAPGKGLEWVAVIWYDGGNKYYADSVKG RFTISRDNSKNTLFLQMNSLRAEDTAVYYCARGGSSWYPDAFDIWGQGTMVTVSSASTKGPSVFPLAPCS RSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 115 24H2 (full length DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWY light chain 1)QQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 116 5F8 (full length EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMH heavy chain)WVRQAPGKGLVWVSRIISDGSSTGYADSVKGRFTI SRDNAKNTLYLQMNSLRAEDTAVYYCAREFSSGWYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 117 5F8 (full length AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWY light chain 1)QQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFSSYPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 118 5F8 (full length DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWY light chain 2)QQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTGFTLTISSLQPEDFATYYCQQYNSYPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 119 6E11 (full length EVQLVESGGALVQPGRSLRLSCAASGFTFDDYAM heavy chain)HWVRQAPGKGLEWVSGITWNSGGIGYADSVKGRF TISRDNAKNSLYLQMNSLRAEDTALYYCAKDRYYSSWLLFDNWGQGILVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 120 6E11 (full length EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAW light chain 1)YQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQHYGSSFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 121 7A11 (full length EVQLVESGGGLVQTGRSLRLSCAASGFTFDDYAM heavy chain)HWVRQAPGKGLEWVSDISWNSDIIGYADSVKGRF TISRDNAKNSLYLQMNSLRAEDTALYYCAKDIYGSGSSFFDYWGQGILVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 122 7A11 (full length DIQMTQSPSSLSASVGDRVTITCRASQYISSWLAWY light chain 1)QQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYHSYPPTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 123 Hinge C219S ERKSCVECPPCPAPPVAG124 IgG2 CH1  ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP (wildtype)VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSNFGTQTYTCNVDHKPSNTKVDKTV 125IgG1 CH2 + PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV A330S and KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT P331SVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAK 126 Human IgG1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP constant regionVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 127 Human IgG1   ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPconstant region VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV (allotypePSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT variant)HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 128 IgG1 CH3 + GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSE356 and M358 DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 129 IgG1 constant ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP regionVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 130 IgG2 constant  ASTKGPSVFP LAPCSRSTSE STAALGCLVKregion DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSNFGTQT YTCNVDHKPSNTKVDKTVER KCCVECP PCPAPPVAG PSVFLFPPKP KDTLMISRTP EVTCVVVDVSHEDPEVQFNW YVDGVEVHNA KTKPREEQFN STFRVVSVLT VVHQDWLNGK EYKCKVSNKGLPAPIEKTIS KTKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQPENNYKTTPPM LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 131Human IgG1  RTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY kappa light PREAKVQWKV DNALQSGNSQ ESVTEQDSKD chain (CL)STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 132 Heavy chain  LSPGKC-terminus 133 CD73.4-IgG2CS- QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMIgG1.1f, AA  HWVRQAPGKGLEWVAVILYDGSNKYYPDSVKGR sequenceFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSS WYPDSFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 134 CD73.4-IgG2CS-caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtcc IgG1.1f, NT ctgagactctcctgtgcagcctctggattcaccttcagtaactatggcatgcactg sequenceggtccgccaggctccaggcaaggggctggagtgggtggcagttatattgtatgatggaagtaataaatactatccagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagagggggcagcagctggtaccctgattcttttgatatctggggccaaggaacaatggtcaccgtctcttcagcgtcgaccaagggcccatcggtcttccccctggcgccctgctccaggagcacctccgagagcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacaccttcccagctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcaacttcggcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagttgagcgcaaatcctgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccaagcagcatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaa 135 CD73.4 VH QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGM (a.a.)HWVRQAPGKGLEWVAVILYDGSNKYYPDSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSSWYPDSFDIWGQGTMVTVSS 136 Wildtype  ERKCCVECPPCPAPPVAG IgG2 hinge 137Wildtype  PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV IgG1 CH2KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK 138Wildtype  GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS IgG1 CH3DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 139 11F11 VH -  CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTG Nucleotide GTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTG SequenceCAACGTCTGGATTCACCTTCAGTAACTATGGCAT GCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATTGTATGATGGAAGT AATAAATACTATCCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGC TGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGGCA GCAGCTGGTACCCTGATTCTTTTGATATCTGGGGCCAAGGAACAATGGTCACCGTCTC TTCA 140 11F11 VK1 - GAAATTGTGTTGACACAGTCTCCAGCCACCCTGT Nucleotide CTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTG SequenceCAGGGCCAGTCAGGGTGTTAGCAGCTACTTAGCC TGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTG GCATCCCAGCCAGGTTCAGTGGCAGTGGGCCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGA GCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCATCTCACTTTCGGCGG AGGGACCAAGGTGGAGATCAAA 141 11F11 VK2 -  GACATCCAGATGACCCAGTCTCCATCCTCACTGT NucleotideCTGCATCTGTAGGAGACAGAGTCACCATCACTTG SequenceTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCC TGGTATCAGCAGAAACCAGAGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTTTGCAAAGTG GGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCA GCCTGAAGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTACCCTCTCACTTTCGGCGG AGGGACCAAGGTGGAGATCAAA 142 4C3 VH -  GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTG NucleotideGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTG SequenceCAGCCTCTGGATTCACCTTTGATGATTATGCCAT GCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAAGAGTGG TAGCATAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCC CTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGTAAAAGGGTATT ACGTTATTTTGACTGGCCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTC A 143 4C3 VK1 - GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGT Nucleotide CTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTG SequenceCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCC TGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTG GCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGA GCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCGCTCACTTTCGGCGGAGGGA CCAAGGTGGAGATCAAA 144 4C3 VK2 - GACATCCAGATGACCCAGTCTCCATCCTCACTGT Nucleotide CTGCATCTGTAGGAGACAGAGTCACCTTCACTTG SequenceTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCC TGGTATCAGCAGAAACCAGAGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTTTGCAAAGTG GGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCA GCCTGAAGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTACCCTCCAACGTTCGGCCA GGGGACCAAGGTGGAAATCAAA 145 4C3 VK3 -  GACATCCAGATGACCCAGTCTCCATCCTCACTGT NucleotideCTGCATCTGTAGGAGACAGAGTCACCTTCACTTG SequenceTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCC TGGTATCAGCAGAAACCAGAGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTTTGCAAAGTG GGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCA GCCTGAAGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTACCCTCCAACGTTCGGCCA AGGGACCAAGGTGGAAATCAAA 146 4D4 VH -  CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTG NucleotideGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTG SequenceCAGCGTCTGGATTCACCTTCAGTAACTATGGCAT GCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGTATGATGAAAG TAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG CTGTTTCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTATTGTGCGAGAGGGTATA ACAGCAGGTGGTACCCTGATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGT CTCTTCA 147 4D4 VK1 -  GACATCCAGATGACCCAGTCTCCATCCTCACTGT NucleotideCTGCATCTGTAGGAGACAGAGTCACCATCACTTG SequenceTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCC TGGTATCAGCAGAAACCAGAGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTTTGCAAAGTG GGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCA GCCTGAAGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTACCCGCTCACTTTCGGCGGAGGGA CCAAGGTGGAGATCAAA 148 10D2 VH1 -  CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTG NucleotideGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTG SequenceCAGCGTCTGGATTCACCTTCAGTAACTATGGCCT GCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATACGGTATGATGGAAG TAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG CTGTATCTGCAAATGAGCAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGGGGGGGC AGCAGCTGGTACCCGGACGGTTTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTC CTCA 149 10D2 VK1 -  GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGT NucleotideCTGCATCTGTAGGAGACAGAGTCACCATCACTTG SequenceCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCC TGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTG GGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCA GCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCCACTTTCGGCGGAGGGACCA AGGTGGAGATCAAA 150 10D2 VK2 -  GACATCCAGATGACCCAGTCTCCATCCTCACTGT NucleotideCTGCATCTGTAGGAGACAGAGTCACCATCACTTG SequenceTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCC TGGTATCAGCAGAAACCAGAGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTTTGCAAAGTG GGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCA GCCTGAAGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTACCCGCTCACTTTCGGCGGAGGGA CCAAGGTGGAGATCAAA 151 11A6 VH - GAAGTGCAGCTGGTGGAATCTGGGGGAAACTTG Nucleotide GTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTG SequenceCAGCCTCTGGATTCACCTTTGATGATTATGCCAT GCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATAATAAT GACATAGGCTATGCGGACTCTGTGAAGGGCCGATTCATCATCTCCAGAGACAACGCCAAGAACTCCC TGTATCTGCAAATGAACAGTCTGAGACCTGAGGACACGGCCTTGTATTATTGTGTAAAAGGTTATTA CGTTATTTTGACTGGTCTTGACTACTGGGGCCAGGGAACCCCGGTCACCGTCTCCTC A 152 11A6 VK1 - GACATCCAGATGACCCAGTCTCCATCCTCACTGT Nucleotide CTGCATCTGTAGGAGACAGAGTCACCATCACTTG SequenceTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCC TGGTATCAGCAGAAACCAGAGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTTTGCAAAGTG GGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCA GCCTGAAGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTACCCGCTCACTTTCGGCGGAGGGA CCAAGGTGGAGATCAAA 153 24H2 VH - CAGGTGCAACTGGTGGAGTCTGGGGGAGGCGTG Nucleotide GTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTG SequenceCAGCGTCTGGATTCACCTTCAGTAACTATGGCAT GCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGTATGATGGAGG TAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG CTGTTTCTGCAAATGAACAGCCTGAGAGCCGAAGACACGGCTGTGTATTACTGTGCGAGAGGGGGC AGCAGCTGGTACCCTGATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTC TTCA 154 24H2 VK1 - GACATCCAGATGACCCAGTCTCCATCCTCACTGT Nucleotide CTGCATCTGTAGGAGACAGAGTCACCATCACTTG SequenceTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCC TGGTATCAGCAGAAACCAGAGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTTTGCAAAGTG GGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCA GCCTGAAGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTACCCTCTCACTTTCGGCGGAGGGA CCAAGGTGGAGATCAAA 155 5F8 VH - GAGGTGCAGCTGGTGGAGTCCGGGGGAGGCTTA Nucleotide GTTCAGCCTGGGGGGTCCCTGAGACTCTCCTGTG SequenceCAGCCTCTGGATTCACCTTCAGTAGCTACTGGAT GCACTGGGTCCGCCAAGCTCCAGGGAAGGGGCTGGTGTGGGTCTCACGTATTATTAGTGATGGGAGT AGCACAGGTTACGCGGATTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACG CTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCAAGAGAGTTTA GCAGTGGCTGGTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA 156 5F8 VK1 -  GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTNucleotide  CTGCATCTGTAGGAGACAGAGTCACCATCACTTG SequenceCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCC TGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTG GGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCA GCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAGTAGTTACCCTCGGACGTTCGGCCAAGGGA CCAAGGTGGAAATCAAA 157 5F8 VK2 - GACATCCAGATGACCCAGTCTCCATCCTCACTGT Nucleotide CTGCATCTGTAGGAGACAGAGTCACCATCACTTG SequenceTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCC TGGTATCAGCAGAAACCAGAGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTTTGCAAAGTG GGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGGTTTCACTCTCACCATCAGCAGCCTGCA GCCTGAAGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTACCCTCGGACGTTCGGCCAAGGGA CCAAGGTGGAAATCAAA 158 6E11 VH - GAAGTGCAGCTGGTGGAGTCTGGGGGAGCCTTG Nucleotide GTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTG SequenceCAGCCTCTGGATTCACCTTTGATGATTATGCCAT GCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTACTTGGAATAGTGGT GGCATAGGCTACGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCC TGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATAGGTA TTACAGCAGTTGGCTCCTCTTTGACAACTGGGGCCAGGGAATTCTGGTCACCGTCTC CTCA 159 6E11 VK1 - GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGT Nucleotide CTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTG SequenceCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTT AGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCC ACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGAC TGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCATTATGGTAGCTCATTCACTTTCGGCCCTGGG ACCAAAGTGGATATCAAA 160 7A11 VH - GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTG Nucleotide GTACAGACTGGCAGGTCCCTGAGACTCTCCTGTG SequenceCAGCCTCTGGATTCACCTTTGATGATTATGCCAT GCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGATATTAGTTGGAATAGTGAT ATTATAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCTAGAGACAACGCCAAGAACTCCCT GTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATATTTAT GGTTCGGGGAGTTCTTTTTTTGACTACTGGGGCCAGGGAATCCTGGTCACCGTCTC CTCA 161 7A11 VK1 - GACATCCAGATGACCCAGTCTCCATCCTCACTGT Nucleotide CTGCATCTGTAGGAGACAGAGTCACCATCACTTG SequenceTCGGGCGAGTCAGTATATTAGCAGCTGGTTAGCC TGGTATCAGCAGAAACCAGAGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTTTGCAAAGTG GGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCA GCCTGAAGATTTTGCAACTTATTACTGCCAACAGTATCATAGTTACCCTCCCACCTTCGGCCA AGGGACACGACTGGAGATTAAA 162 IgG1-IgG2-ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP IgG1f2 (MHCCR)VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSSLGTQTYICNVNHKPSNTKVDKKVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 163 IgG1-IgG2CS- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPIgG1f2 (MHCCR) VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVERKSCVEC PPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK 164 IgG2-IgG1f2 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP (MHCCR)VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 165 IgG2CS-IgG1f2  ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP(MHCCR) VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVEC PPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK 166 IgG1-IgG2-ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP IgG1.1f (MHCCR)VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSSLGTQTYICNVNHKPSNTKVDKKVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPS SIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 167 IgG1-IgG2CS- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPIgG1.1f (MHCCR) VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVERKSCVEC PPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK 168 IgG2-IgG1.1f ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP (MHCCR)VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPS SIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 169 IgG2CS-IgG1.1f  ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP(MHCCR) VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVEC PPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK 170 CD73.3 VH (a.a)EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAM HWVRQAPGKGLEWVSGISWKSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTVLYYCVKGYYVI LTGLDYWGQGTLVTVSS 171CD73.5 VH (a.a) QVQLVESGGGVVQPGRSLRLSCASSGFTFSNYGMHWVRQAPGKGLEWVAVILYDGSNKYYPDSVKGRFT ISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSSWYPDSFDIWGQGTMVTVSS 172 CD73.6 VH (a.a)QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGM HWVRQAPGKGLEWVAVILYDSSNKYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSS WYPDSFDIWGQGTMVTVSS 173CD73.7 VH (a.a) QVQLVESGGGVVQPGRSLRLSCASSGFTFSNYGMHWVRQAPGKGLEWVAVILYDSSNKYYPDSVKGRFT ISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSSWYPDSFDIWGQGTMVTVSS 174 CD73.8 VH (a.a)QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGM HWVRQAPGKGLEWVAVIWYDSSNKYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSS WYPDSFDIWGQGTMVTVSS 175CD73.9 VH (a.a) QVQLVESGGGVVQPGRSLRLSCASSGFTFSNYGMHWVRQAPGKGLEWVAVIWYDSSNKYYPDSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSSWYPDSFDIWGQGTMVTVSS 176 CD73.10 VH (a.a)QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGM HWVRQAPGKGLEWVAVIWYDESNKYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSS WYPDSFDIWGQGTMVTVSS 177CD73.11 VH (a.a) QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVIWYDESNKYYADSVKGR FTISRDNSKNTLFLQMNSLRAEDTAVYYCARGYNSRWYPDAFDIWGQGTMVTVSS 178 IgG2/IgG1 hybrid  ERKCCVECPPCPAPELLGG hinge 179IgG2 C219S/IgG1  ERKSCVECPPCPAPELLGG hybrid hinge 180 IgG1-IgG2-IgG1fASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVERKCCVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 181 IgG1-IgG2CS-IgG1f ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSSLGTQTYICNVNHKPSNTKVDKKVERKSCVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 182 IgG2-IgG1f ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 183 IgG2CS-IgG1f ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 184 mAb-CD73.3-Vh- EVQLVESGGG LVQPGRSLRL SCAASGFTFDhHC-IgG1.1f DYAMHWVRQA PGKGLEWVSGISWKSGSIGYADSVKGRFTI SRDNAKNSLY LQMNSLRAED TALYYCAKGYYVILTGLDYW GQGTLVTVSSASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSSGLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPEAEGAPSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYNSTYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPSSIEKTIS KAKGQPREPQ VYTLPPSREEMTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRWQQGNVFSCSV MHEALHNHYT QKSLSLSPG 185 mAb-CD73.3-Vh-EVQLVESGGG LVQPGRSLRL SCAASGFTFD hHC-IgG2-C219SDYAMHWVRQA PGKGLEWVSG ISWKSGSIGY ADSVKGRFTI SRDNAKNSLY LQMNSLRAEDTALYYCAKGY YVILTGLDYW GQGTLVTVSS ASTKGPSVFP LAPCSRSTSE STAALGCLVKDYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSNFGTQT YTCNVDHKPSNTKVDKTVER KSCVECPPCP APPVAGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDPEVQFNWYVDG VEVHNAKTKP REEQFNSTFR VVSVLTVVHQ DWLNGKEYKC KVSNKGLPAPIEKTISKTKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNYKTTPPMLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPG 186mAb-CD73.3-Vh- EVQLVESGGG LVQPGRSLRL SCAASGFTFD hHC-IgG2-C219S-DYAMHWVRQA PGKGLEWVSG ISWKSGSIGY IgG1.1fADSVKGRFTI SRDNAKNSLY LQMNSLRAED TALYYCAKGY YVILTGLDYW GQGTLVTVSSASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSSGLYSLSSVVT VPSSNFGTQT YTCNVDHKPS NTKVDKTVER KSCVECPPCP APPVAGPSVFLFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYRVVSVLTVLHQ DWLNGKEYKC KVSNKALPSS IEKTISKAKG QPREPQVYTL PPSREEMTKNQVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGNVFSCSVMHEA LHNHYTQKSL SLSPG 187 mAb-CD73.4-Vh-QVQLVESGGG VVQPGRSLRL SCAASGFTFS hHC-IgG1.1fNYGMHWVRQA PGKGLEWVAV ILYDGSNKYY PDSVKGRFTI SRDNSKNTLY LQMNSLRAEDTAVYYCARGG SSWYPDSFDI WGQGTMVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLVKDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTQ TYICNVNHKPSNTKVDKRVE PKSCDKTHTC PPCPAPEAEG APSVFLFPPK PKDTLMISRT PEVTCVVVDVSHEDPEVKFN WYVDGVEVHN AKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNKALPSSIEKTI SKAKGQPREP QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD IAVEWESNGQPENNYKTTPP VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG 188mAb-CD73.4-Vh- QVQLVESGGG VVQPGRSLRL SCAASGFTFS hHC-IgG2-C219SNYGMHWVRQA PGKGLEWVAV ILYDGSNKYY PDSVKGRFTI SRDNSKNTLY LQMNSLRAEDTAVYYCARGG SSWYPDSFDI WGQGTMVTVS SASTKGPSVF PLAPCSRSTS ESTAALGCLVKDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSNFGTQ TYTCNVDHKPSNTKVDKTVE RKSCVECPPC PAPPVAGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHEDPEVQFNWYVD GVEVHNAKTK PREEQFNSTF RVVSVLTVVH QDWLNGKEYK CKVSNKGLPAPIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENNYKTTPPMLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG 189mAb-CD73.4-Vh- QVQLVESGGG VVQPGRSLRL SCAASGFTFS hHC-IgG2-C219S-NYGMHWVRQA PGKGLEWVAV ILYDGSNKYY IgG1.1fPDSVKGRFTI SRDNSKNTLY LQMNSLRAED (identical to TAVYYCARGG SSWYPDSFDI WGQGTMVTVS SEQ ID NO: 133,SASTKGPSVF PLAPCSRSTS ESTAALGCLV except lacks C-KDYFPEPVTV SWNSGALTSG VHTFPAVLQS terminal lysine)SGLYSLSSVV TVPSSNFGTQ TYTCNVDHKP SNTKVDKTVE RKSCVECPPC PAPPVAGPSVFLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTYRVVSVLTVLH QDWLNGKEYK CKVSNKALPS SIEKTISKAK GQPREPQVYT LPPSREEMTKNQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQGNVFSCSVMHE ALHNHYTQKS LSLSPG 190 mAb-CD73.5-Vh-QVQLVESGGG VVQPGRSLRL SCASSGFTFS hHC-IgG1.1fNYGMHWVRQA PGKGLEWVAV ILYDGSNKYY PDSVKGRFTI SRDNSKNTLY LQMNSLRAEDTAVYYCARGG SSWYPDSFDI WGQGTMVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLVKDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTQ TYICNVNHKPSNTKVDKRVE PKSCDKTHTC PPCPAPEAEG APSVFLFPPK PKDTLMISRT PEVTCVVVDVSHEDPEVKFN WYVDGVEVHN AKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNKALPSSIEKTI SKAKGQPREP QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD IAVEWESNGQPENNYKTTPP VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG 191mAb-CD73.5-Vh- QVQLVESGGG VVQPGRSLRL SCASSGFTFS hHC-IgG2-C219SNYGMHWVRQA PGKGLEWVAV ILYDGSNKYY PDSVKGRFTI SRDNSKNTLY LQMNSLRAEDTAVYYCARGG SSWYPDSFDI WGQGTMVTVS SASTKGPSVF PLAPCSRSTS ESTAALGCLVKDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSNFGTQ TYTCNVDHKPSNTKVDKTVE RKSCVECPPC PAPPVAGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHEDPEVQFNWYVD GVEVHNAKTK PREEQFNSTF RVVSVLTVVH QDWLNGKEYK CKVSNKGLPAPIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENNYKTTPPMLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG 192mAb-CD73.5-Vh- QVQLVESGGG VVQPGRSLRL SCASSGFTFS hHC-IgG2-C219S-NYGMHWVRQA PGKGLEWVAV ILYDGSNKYY IgG1.1fPDSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG SSWYPDSFDI WGQGTMVTVSSASTKGPSVF PLAPCSRSTS ESTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQSSGLYSLSSVV TVPSSNFGTQ TYTCNVDHKP SNTKVDKTVE RKSCVECPPC PAPPVAGPSVFLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTYRVVSVLTVLH QDWLNGKEYK CKVSNKALPS SIEKTISKAK GQPREPQVYT LPPSREEMTKNQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQGNVFSCSVMHE ALHNHYTQKS LSLSPG 193 mAb-CD73.6-Vh-QVQLVESGGG VVQPGRSLRL SCAASGFTFS hHC-IgG1.1fNYGMHWVRQA PGKGLEWVAV ILYDSSNKYY PDSVKGRFTI SRDNSKNTLY LQMNSLRAEDTAVYYCARGG SSWYPDSFDI WGQGTMVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLVKDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTQ TYICNVNHKPSNTKVDKRVE PKSCDKTHTC PPCPAPEAEG APSVFLFPPK PKDTLMISRT PEVTCVVVDVSHEDPEVKFN WYVDGVEVHN AKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNKALPSSIEKTI SKAKGQPREP QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD IAVEWESNGQPENNYKTTPP VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG 194mAb-CD73.6-Vh- QVQLVESGGG VVQPGRSLRL SCAASGFTFS hHC-IgG2-C219SNYGMHWVRQA PGKGLEWVAV ILYDSSNKYY PDSVKGRFTI SRDNSKNTLY LQMNSLRAEDTAVYYCARGG SSWYPDSFDI WGQGTMVTVS SASTKGPSVF PLAPCSRSTS ESTAALGCLVKDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSNFGTQ TYTCNVDHKPSNTKVDKTVE RKSCVECPPC PAPPVAGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHEDPEVQFNWYVD GVEVHNAKTK PREEQFNSTF RVVSVLTVVH QDWLNGKEYK CKVSNKGLPAPIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENNYKTTPPMLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG 195mAb-CD73.6-Vh- QVQLVESGGG VVQPGRSLRL SCAASGFTFS hHC-IgG2-C219S-NYGMHWVRQA PGKGLEWVAV ILYDSSNKYY IgG1.1fPDSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG SSWYPDSFDI WGQGTMVTVSSASTKGPSVF PLAPCSRSTS ESTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQSSGLYSLSSVV TVPSSNFGTQ TYTCNVDHKP SNTKVDKTVE RKSCVECPPC PAPPVAGPSVFLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTYRVVSVLTVLH QDWLNGKEYK CKVSNKALPS SIEKTISKAK GQPREPQVYT LPPSREEMTKNQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQGNVFSCSVMHE ALHNHYTQKS LSLSPGK 196 mAb-CD73.7-Vh-QVQLVESGGG VVQPGRSLRL SCASSGFTFS hHC-IgG1.1fNYGMHWVRQA PGKGLEWVAV ILYDSSNKYY PDSVKGRFTI SRDNSKNTLY LQMNSLRAEDTAVYYCARGG SSWYPDSFDI WGQGTMVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLVKDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTQ TYICNVNHKPSNTKVDKRVE PKSCDKTHTC PPCPAPEAEG APSVFLFPPK PKDTLMISRT PEVTCVVVDVSHEDPEVKFN WYVDGVEVHN AKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNKALPSSIEKTI SKAKGQPREP QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD IAVEWESNGQPENNYKTTPP VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG 197mAb-CD73.7-Vh- QVQLVESGGG VVQPGRSLRL SCASSGFTFS hHC-IgG2-C219SNYGMHWVRQA PGKGLEWVAV ILYDSSNKYY PDSVKGRFTI SRDNSKNTLY LQMNSLRAEDTAVYYCARGG SSWYPDSFDI WGQGTMVTVS SASTKGPSVF PLAPCSRSTS ESTAALGCLVKDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSNFGTQ TYTCNVDHKPSNTKVDKTVE RKSCVECPPC PAPPVAGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHEDPEVQFNWYVD GVEVHNAKTK PREEQFNSTF RVVSVLTVVH QDWLNGKEYK CKVSNKGLPAPIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENNYKTTPPMLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG 198mAb-CD73.7-Vh- QVQLVESGGG VVQPGRSLRL SCASSGFTFS hHC-IgG2-C219S-NYGMHWVRQA PGKGLEWVAV ILYDSSNKYY IgG1.1fPDSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG SSWYPDSFDI WGQGTMVTVSSASTKGPSVF PLAPCSRSTS ESTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQSSGLYSLSSVV TVPSSNFGTQ TYTCNVDHKP SNTKVDKTVE RKSCVECPPC PAPPVAGPSVFLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTYRVVSVLTVLH QDWLNGKEYK CKVSNKALPS SIEKTISKAK GQPREPQVYT LPPSREEMTKNQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQGNVFSCSVMHE ALHNHYTQKS LSLSPG 199 mAb-CD73.8-Vh-QVQLVESGGG VVQPGRSLRL SCAASGFTFS hHC-IgG1.1fNYGMHWVRQA PGKGLEWVAV IWYDSSNKYY PDSVKGRFTI SRDNSKNTLY LQMNSLRAEDTAVYYCARGG SSWYPDSFDI WGQGTMVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLVKDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTQ TYICNVNHKPSNTKVDKRVE PKSCDKTHTC PPCPAPEAEG APSVFLFPPK PKDTLMISRT PEVTCVVVDVSHEDPEVKFN WYVDGVEVHN AKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNKALPSSIEKTI SKAKGQPREP QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD IAVEWESNGQPENNYKTTPP VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG 200mAb-CD73.8-Vh- QVQLVESGGG VVQPGRSLRL SCAASGFTFS hHC-IgG2-C219SNYGMHWVRQA PGKGLEWVAV IWYDSSNKYY PDSVKGRFTI SRDNSKNTLY LQMNSLRAEDTAVYYCARGG SSWYPDSFDI WGQGTMVTVS SASTKGPSVF PLAPCSRSTS ESTAALGCLVKDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSNFGTQ TYTCNVDHKPSNTKVDKTVE RKSCVECPPC PAPPVAGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHEDPEVQFNWYVD GVEVHNAKTK PREEQFNSTF RVVSVLTVVH QDWLNGKEYK CKVSNKGLPAPIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENNYKTTPPMLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG 201mAb-CD73.8-Vh- QVQLVESGGG VVQPGRSLRL SCAASGFTFS hHC-IgG2-C219S-NYGMHWVRQA PGKGLEWVAV IWYDSSNKYY IgG1.1fPDSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG SSWYPDSFDI WGQGTMVTVSSASTKGPSVF PLAPCSRSTS ESTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQSSGLYSLSSVV TVPSSNFGTQ TYTCNVDHKP SNTKVDKTVE RKSCVECPPC PAPPVAGPSVFLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTYRVVSVLTVLH QDWLNGKEYK CKVSNKALPS SIEKTISKAK GQPREPQVYT LPPSREEMTKNQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQGNVFSCSVMHE ALHNHYTQKS LSLSPG 202 mAb-CD73.9-Vh-QVQLVESGGG VVQPGRSLRL SCASSGFTFS hHC-IgG1.1fNYGMHWVRQA PGKGLEWVAV IWYDSSNKYY PDSVKGRFTI SRDNSKNTLY LQMNSLRAEDTAVYYCARGG SSWYPDSFDI WGQGTMVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLVKDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTQ TYICNVNHKPSNTKVDKRVE PKSCDKTHTC PPCPAPEAEG APSVFLFPPK PKDTLMISRT PEVTCVVVDVSHEDPEVKFN WYVDGVEVHN AKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNKALPSSIEKTI SKAKGQPREP QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD IAVEWESNGQPENNYKTTPP VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG 203mAb-CD73.9-Vh- QVQLVESGGG VVQPGRSLRL SCASSGFTFS hHC-IgG2-C219SNYGMHWVRQA PGKGLEWVAV IWYDSSNKYY PDSVKGRFTI SRDNSKNTLY LQMNSLRAEDTAVYYCARGG SSWYPDSFDI WGQGTMVTVS SASTKGPSVF PLAPCSRSTS ESTAALGCLVKDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSNFGTQ TYTCNVDHKPSNTKVDKTVE RKSCVECPPC PAPPVAGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHEDPEVQFNWYVD GVEVHNAKTK PREEQFNSTF RVVSVLTVVH QDWLNGKEYK CKVSNKGLPAPIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENNYKTTPPMLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG 204mAb-CD73.9-Vh- QVQLVESGGG VVQPGRSLRL SCASSGFTFS hHC-IgG2-C219S-NYGMHWVRQA PGKGLEWVAV IWYDSSNKYY IgG1.1fPDSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG SSWYPDSFDI WGQGTMVTVSSASTKGPSVF PLAPCSRSTS ESTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQSSGLYSLSSVV TVPSSNFGTQ TYTCNVDHKP SNTKVDKTVE RKSCVECPPC PAPPVAGPSVFLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTYRVVSVLTVLH QDWLNGKEYK CKVSNKALPS SIEKTISKAK GQPREPQVYT LPPSREEMTKNQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQGNVFSCSVMHE ALHNHYTQKS LSLSPG 205 mAb-CD73.10-Vh-QVQLVESGGG VVQPGRSLRL SCAASGFTFS hHC-IgG1.1fNYGMHWVRQA PGKGLEWVAV IWYDESNKYY PDSVKGRFTI SRDNSKNTLY LQMNSLRAEDTAVYYCARGG SSWYPDSFDI WGQGTMVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLVKDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTQ TYICNVNHKPSNTKVDKRVE PKSCDKTHTC PPCPAPEAEG APSVFLFPPK PKDTLMISRT PEVTCVVVDVSHEDPEVKFN WYVDGVEVHN AKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNKALPSSIEKTI SKAKGQPREP QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD IAVEWESNGQPENNYKTTPP VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG 206mAb-CD73.10-Vh- QVQLVESGGG VVQPGRSLRL SCAASGFTFS hHC-IgG2-C219SNYGMHWVRQA PGKGLEWVAV IWYDESNKYY PDSVKGRFTI SRDNSKNTLY LQMNSLRAEDTAVYYCARGG SSWYPDSFDI WGQGTMVTVS SASTKGPSVF PLAPCSRSTS ESTAALGCLVKDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSNFGTQ TYTCNVDHKPSNTKVDKTVE RKSCVECPPC PAPPVAGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHEDPEVQFNWYVD GVEVHNAKTK PREEQFNSTF RVVSVLTVVH QDWLNGKEYK CKVSNKGLPAPIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENNYKTTPPMLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG 207mAb-CD73.10-Vh- QVQLVESGGG VVQPGRSLRL SCAASGFTFS hHC-IgG2-C219S-NYGMHWVRQA PGKGLEWVAV IWYDESNKYY IgG1.1fPDSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG SSWYPDSFDI WGQGTMVTVSSASTKGPSVF PLAPCSRSTS ESTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQSSGLYSLSSVV TVPSSNFGTQ TYTCNVDHKP SNTKVDKTVE RKSCVECPPC PAPPVAGPSVFLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTYRVVSVLTVLH QDWLNGKEYK CKVSNKALPS SIEKTISKAK GQPREPQVYT LPPSREEMTKNQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQGNVFSCSVMHE ALHNHYTQKS LSLSPG 208 mAb-CD73.11-Vh-QVQLVESGGG VVQPGRSLRL SCAASGFTFS hHC-IgG1.1fNYGMHWVRQA PGKGLEWVAV IWYDESNKYY ADSVKGRFTI SRDNSKNTLF LQMNSLRAEDTAVYYCARGY NSRWYPDAFD IWGQGTMVTV SSASTKGPSV FPLAPSSKST SGGTAALGCLVKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT QTYICNVNHKPSNTKVDKRV EPKSCDKTHT CPPCPAPEAE GAPSVFLFPP KPKDTLMISR TPEVTCVVVDVSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSNKALPSSIEKT ISKAKGQPRE PQVYTLPPSR EEMTKNQVSL TCLVKGFYPS DIAVEWESNGQPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSPG 209mAb-CD73.11-Vh- QVQLVESGGG VVQPGRSLRL SCAASGFTFS hHC-IgG2-C219SNYGMHWVRQA PGKGLEWVAV IWYDESNKYY ADSVKGRFTI SRDNSKNTLF LQMNSLRAEDTAVYYCARGY NSRWYPDAFD IWGQGTMVTV SSASTKGPSV FPLAPCSRST SESTAALGCLVKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSNFGT QTYTCNVDHKPSNTKVDKTV ERKSCVECPP CPAPPVAGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHEDPEVQFNWYV DGVEVHNAKT KPREEQFNST FRVVSVLTVV HQDWLNGKEY KCKVSNKGLPAPIEKTISKT KGQPREPQVY TLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPENNYKTTPPMLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPG 210mAb-CD73.11-Vh- QVQLVESGGG VVQPGRSLRL SCAASGFTFS hHC-IgG2-C219S-NYGMHWVRQA PGKGLEWVAV IWYDESNKYY IgG1.1fADSVKGRFTI SRDNSKNTLF LQMNSLRAED TAVYYCARGY NSRWYPDAFD IWGQGTMVTVSSASTKGPSV FPLAPCSRST SESTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQSSGLYSLSSV VTVPSSNFGT QTYTCNVDHK PSNTKVDKTV ERKSCVECPP CPAPPVAGPSVFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNSTYRVVSVLTVL HQDWLNGKEY KCKVSNKALP SSIEKTISKA KGQPREPQVY TLPPSREEMTKNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQGNVFSCSVMH EALHNHYTQK SLSLSPG 211 mAb-CD73.3-Vh-gaagtgcagctggtggagtctgggggaggcttggtacagcctggcaggtccct hHC-IgG1.1fgagactctcctgtgcagcctctggattcacattgatgattatgccatgcactgggtccggcaagctccagggaagggcctggagtgggtctcaggtattagttggaagagtggtagcataggctatgcggactctgtgaagggccgattcaccatctccagagacaacgccaagaactccctgtatctgcaaatgaacagtctgagagctgaggacacggccttgtattactgtgccaaagggtattacgttattttgactggccttgactactggggccagggaaccctggtcaccgtctcctcagcgtcgaccaagggcccctccgtgtttcctctggccccttccagcaagtccacctctggcggaacagccgctctgggctgcctggtcaaggactacttccccgagcctgtgaccgtgtcctggaactctggcgccctgacatctggcgtgcacaccttccctgctgtgctgcagtctagcggcctgtactccctgtcctccgtcgtgacagtgccctccagctctctgggcacccagacctacatctgcaacgtgaaccacaagccctccaacaccaaggtggacaagcgggtggaacccaagtcctgcgacaagacccatacctgccctccctgccctgctcctgaagctgaaggcgcccctagcgtgttcctgttccctccaaagcccaaggacaccctgatgatctcccggacccctgaagtgacctgcgtggtggtggatgtgtcccacgaggacccagaagtgaagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcctagagaggaacagtacaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggattggctgaacggcaaagagtacaagtgcaaggtgtccaacaaggccctgcctagctccatcgaaaagaccatctccaaggctaagggccagccccgcgagccccaggtgtacacactgcctccatcccgggaagagatgaccaagaaccaggtgtccctgacttgcctcgtgaagggcttctacccctccgatatcgccgtggaatgggagtccaacggccagcctgagaacaactacaagaccacccctcccgtgctggactccgacggctcattcttcctgtacagcaagctgacagtggataagtcccggtggcagcaggggaacgtgttctcctgctccgtgatgcacgaggctctgcacaaccactacacacagaagtccctgtctctgtcccctggc 212 mAb-CD73.3-Vh-gaagtgcagctggtggagtctgggggaggcttggtacagcctggcaggtccct hHC-IgG2-C219Sgagactctcctgtgcagcctctggattcacattgatgattatgccatgcactgggtccggcaagctccagggaagggcctggagtgggtctcaggtattagttggaagagtggtagcataggctatgcggactctgtgaagggccgattcaccatctccagagacaacgccaagaactccctgtatctgcaaatgaacagtctgagagctgaggacacggccttgtattactgtgccaaagggtattacgttattttgactggccttgactactggggccagggaaccctggtcaccgtctcctcagcgtcgaccaagggcccctctgtgtttcctctggccccttgctcccggtccacctctgagtctaccgctgctctgggctgcctggtcaaggactacttccccgagcctgtgaccgtgtcctggaactctggcgctctgacctccggcgtgcacacattccagccgtgctgcagtcctccggcctgtactctctgtcctccgtcgtgaccgtgccctcctccaacttcggcacccagacctacacctgtaacgtggaccacaagccctccaacaccaaggtggacaagaccgtggaacggaagtcctgcgtggaatgccctccttgccctgcacctcctgtggctggcccttccgtgttcctgttccccccaaagcccaaggacaccctgatgatctcccggacccccgaagtgacctgcgtggtggtggatgtgtcccacgaggaccccgaggtgcagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagttcaactccaccttccgggtggtgtccgtgctgaccgtggtgcaccaggactggctgaacggcaaagagtacaagtgcaaggtgtccaacaagggcctgcctgcccccatcgaaaagaccatctccaagacaaagggccagccccgcgagcctcaggtgtacacactgcctcccagccgggaagagatgaccaagaaccaggtgtccctgacctgtctggtcaagggcttctacccctccgatatcgccgtggaatgggagtccaacggccagcccgagaacaactacaagaccaccccccccatgctggactccgacggctcattcttcctgtactccaagctgacagtggacaagtcccggtggcagcagggcaacgtgttctcctgctctgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagccccggcaa 213 mAb-CD73.3-Vh-gaagtgcagctggtggagtctgggggaggcttggtacagcctggcaggtccct hHC-IgG2-C219S-gagactctcctgtgcagcctctggattcacattgatgattatgccatgcactgggt IgG1.1fccggcaagctccagggaagggcctggagtgggtctcaggtattagttggaagagtggtagcataggctatgcggactctgtgaagggccgattcaccatctccagagacaacgccaagaactccctgtatctgcaaatgaacagtctgagagctgaggacacggccttgtattactgtgccaaagggtattacgttattttgactggccttgactactggggccagggaaccctggtcaccgtctcctcagcgtcgaccaagggcccatcggtcttccccctggcgccctgctccaggagcacctccgagagcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacaccttcccagctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcaacttcggcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagttgagcgcaaatcctgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccaagcagcatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaa 214 mAb-CD73.4-Vh-caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccc hHC-IgG1.1ftgagactctcctgtgcagcctctggattcaccttcagtaactatggcatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatattgtatgatggaagtaataaatactatccagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagagggggcagcagctggtaccctgattcttttgatatctggggccaaggaacaatggtcaccgtctcttcagcgtcgaccaagggcccctccgtgtttcctctggccccttccagcaagtccacctctggcggaacagccgctctgggctgcctggtcaaggactacttccccgagcctgtgaccgtgtcctggaactctggcgccctgacatctggcgtgcacaccttccctgctgtgctgcagtctagcggcctgtactccctgtcctccgtcgtgacagtgccctccagctctctgggcacccagacctacatctgcaacgtgaaccacaagccctccaacaccaaggtggacaagcgggtggaacccaagtcctgcgacaagacccatacctgccctccctgccctgctcctgaagctgaaggcgcccctagcgtgttcctgttccctccaaagcccaaggacaccctgatgatctcccggacccctgaagtgacctgcgtggtggtggatgtgtcccacgaggacccagaagtgaagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcctagagaggaacagtacaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggattggctgaacggcaaagagtacaagtgcaaggtgtccaacaaggccctgcctagctccatcgaaaagaccatctccaaggctaagggccagccccgcgagccccaggtgtacacactgcctccatcccgggaagagatgaccaagaaccaggtgtccctgacttgcctcgtgaagggcttctacccctccgatatcgccgtggaatgggagtccaacggccagcctgagaacaactacaagaccacccctcccgtgctggactccgacggctcattcttcctgtacagcaagctgacagtggataagtcccggtggcagcaggggaacgtgttctcctgctccgtgatgcacgaggctctgcacaaccactacacacagaagtccctgtctctgtcccctggc 215 mAb-CD73.4-Vh-caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccc hHC-IgG2-C219Stgagactctcctgtgcagcctctggattcaccttcagtaactatggcatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatattgtatgatggaagtaataaatactatccagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagagggggcagcagctggtaccctgattcttttgatatctggggccaaggaacaatggtcaccgtctcttcagcgtcgaccaagggcccctctgtgtttcctctggccccttgctcccggtccacctctgagtctaccgctgctctgggctgcctggtcaaggactacttccccgagcctgtgaccgtgtcctggaactctggcgctctgacctccggcgtgcacacctttccagccgtgctgcagtcctccggcctgtactctctgtcctccgtcgtgaccgtgccctcctccaacttcggcacccagacctacacctgtaacgtggaccacaagccctccaacaccaaggtggacaagaccgtggaacggaagtcctgcgtggaatgccctccttgccctgcacctcctgtggctggcccttccgtgttcctgttccccccaaagcccaaggacaccctgatgatctcccggacccccgaagtgacctgcgtggtggtggatgtgtcccacgaggaccccgaggtgcagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagttcaactccaccttccgggtggtgtccgtgctgaccgtggtgcaccaggactggctgaacggcaaagagtacaagtgcaaggtgtccaacaagggcctgcctgcccccatcgaaaagaccatctccaagacaaagggccagccccgcgagcctcaggtgtacacactgcctcccagccgggaagagatgaccaagaaccaggtgtccctgacctgtctggtcaagggcttctacccctccgatatcgccgtggaatgggagtccaacggccagcccgagaacaactacaagaccaccccccccatgctggactccgacggctcattcttcctgtactccaagctgacagtggacaagtcccggtggcagcagggcaacgtgttctcctgctctgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagccccggcaaa 216 mAb-CD73.4-Vh-caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccc hHC-IgG2-C219S-tgagactctcctgtgcagcctctggattcaccttcagtaactatggcatgcactgg IgG1.1fgtccgccaggctccaggcaaggggctggagtgggtggcagttatattgtatgatggaagtaataaatactatccagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagagggggcagcagctggtaccctgattcttttgatatctggggccaaggaacaatggtcaccgtctcttcagcgtcgaccaagggcccatcggtcttccccctggcgccctgctccaggagcacctccgagagcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacaccttcccagctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcaacttcggcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagttgagcgcaaatcctgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccaagcagcatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaa 217 mAb-CD73.5-Vh-caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccc hHC-IgG1.1ftgagactctcctgtgcaagctctggattcaccttcagtaactatggcatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatattgtatgatggaagtaataaatactatccagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagagggggcagcagctggtaccctgattcttttgatatctggggccaaggaacaatggtcaccgtctcttcagcgtcgaccaagggcccctccgtgtttcctctggccccttccagcaagtccacctctggcggaacagccgctctgggctgcctggtcaaggactacttccccgagcctgtgaccgtgtcctggaactctggcgccctgacatctggcgtgcacaccttccctgctgtgctgcagtctagcggcctgtactccctgtcctccgtcgtgacagtgccctccagctctctgggcacccagacctacatctgcaacgtgaaccacaagccctccaacaccaaggtggacaagcgggtggaacccaagtcctgcgacaagacccatacctgccctccctgccctgctcctgaagctgaaggcgcccctagcgtgttcctgttccctccaaagcccaaggacaccctgatgatctcccggacccctgaagtgacctgcgtggtggtggatgtgtcccacgaggacccagaagtgaagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcctagagaggaacagtacaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggattggctgaacggcaaagagtacaagtgcaaggtgtccaacaaggccctgcctagctccatcgaaaagaccatctccaaggctaagggccagccccgcgagccccaggtgtacacactgcctccatcccgggaagagatgaccaagaaccaggtgtccctgacttgcctcgtgaagggcttctacccctccgatatcgccgtggaatgggagtccaacggccagcctgagaacaactacaagaccacccctcccgtgctggactccgacggctcattcttcctgtacagcaagctgacagtggataagtcccggtggcagcaggggaacgtgttctcctgctccgtgatgcacgaggctctgcacaaccactacacacagaagtccctgtctctgtcccctggc 218 mAb-CD73.5-Vh-caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccc hHC-IgG2-C219Stgagactctcctgtgcaagctctggattcaccttcagtaactatggcatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatattgtatgatggaagtaataaatactatccagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagagggggcagcagctggtaccctgattcttttgatatctggggccaaggaacaatggtcaccgtctcttcagcgtcgaccaagggcccctctgtgtttcctctggccccttgctcccggtccacctctgagtctaccgctgctctgggctgcctggtcaaggactacttccccgagcctgtgaccgtgtcctggaactctggcgctctgacctccggcgtgcacacattccagccgtgctgcagtcctccggcctgtactctctgtcctccgtcgtgaccgtgccctcctccaacttcggcacccagacctacacctgtaacgtggaccacaagccctccaacaccaaggtggacaagaccgtggaacggaagtcctgcgtggaatgccctccttgccctgcacctcctgtggctggcccttccgtgttcctgttccccccaaagcccaaggacaccctgatgatctcccggacccccgaagtgacctgcgtggtggtggatgtgtcccacgaggaccccgaggtgcagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagttcaactccaccttccgggtggtgtccgtgctgaccgtggtgcaccaggactggctgaacggcaaagagtacaagtgcaaggtgtccaacaagggcctgcctgcccccatcgaaaagaccatctccaagacaaagggccagccccgcgagcctcaggtgtacacactgcctcccagccgggaagagatgaccaagaaccaggtgtccctgacctgtctggtcaagggcttctacccctccgatatcgccgtggaatgggagtccaacggccagcccgagaacaactacaagaccaccccccccatgctggactccgacggctcattcttcctgtactccaagctgacagtggacaagtcccggtggcagcagggcaacgtgttctcctgctctgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagccccggcaaa 219 mAb-CD73.5-Vh-caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccc hHC-IgG2-C219S-tgagactctcctgtgcaagctctggattcaccttcagtaactatggcatgcactgg IgG1.1fgtccgccaggctccaggcaaggggctggagtgggtggcagttatattgtatgatggaagtaataaatactatccagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagagggggcagcagctggtaccctgattcttttgatatctggggccaaggaacaatggtcaccgtctcttcagcgtcgaccaagggcccatcggtcttccccctggcgccctgctccaggagcacctccgagagcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacaccttcccagctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcaacttcggcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagttgagcgcaaatcctgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccaagcagcatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaa 220 mAb-CD73.6-Vh-ggcatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcag hHC-IgG1.1fttatattgtatgattccagtaataaatactatccagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagagggggcagcagctggtaccctgattcttttgatatctggggccaaggaacaatggtcaccgtctcttcagcgtcgaccaagggcccctccgtgtttcctctggccccttccagcaagtccacctctggcggaacagccgctctgggctgcctggtcaaggactacttccccgagcctgtgaccgtgtcctggaactctggcgccctgacatctggcgtgcacaccttccctgctgtgctgcagtctagcggcctgtactccctgtcctccgtcgtgacagtgccctccagctctctgggcacccagacctacatctgcaacgtgaaccacaagccctccaacaccaaggtggacaagcgggtggaacccaagtcctgcgacaagacccatacctgccctccctgccctgctcctgaagctgaaggcgcccctagcgtgttcctgttccctccaaagcccaaggacaccctgatgatctcccggacccctgaagtgacctgcgtggtggtggatgtgtcccacgaggacccagaagtgaagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcctagagaggaacagtacaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggattggctgaacggcaaagagtacaagtgcaaggtgtccaacaaggccctgcctagctccatcgaaaagaccatctccaaggctaagggccagccccgcgagccccaggtgtacacactgcctccatcccgggaagagatgaccaagaaccaggtgtccctgacttgcctcgtgaagggcttctacccctccgatatcgccgtggaatgggagtccaacggccagcctgagaacaactacaagaccacccctcccgtgctggactccgacggctcattcttcctgtacagcaagctgacagtggataagtcccggtggcagcaggggaacgtgttctcctgctccgtgatgcacgaggctctgcacaaccactacacacagaagtccctgtctctgtc ccctggc 221mAb-CD73.6-Vh- caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtcccthHC-IgG2-C219S gagactctcctgtgcagcctctggattcaccttcagtaactatggcatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatattgtatgattccagtaataaatactatccagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagagggggcagcagctggtaccctgattcttttgatatctggggccaaggaacaatggtcaccgtctcttcagcgtcgaccaagggcccctctgtgtttcctctggccccttgctcccggtccacctctgagtctaccgctgctctgggctgcctggtcaaggactacttccccgagcctgtgaccgtgtcctggaactctggcgctctgacctccggcgtgcacacctttccagccgtgctgcagtcctccggcctgtactctctgtcctccgtcgtgaccgtgccctcctccaacttcggcacccagacctacacctgtaacgtggaccacaagccctccaacaccaaggtggacaagaccgtggaacggaagtcctgcgtggaatgccctccttgccctgcacctcctgtggctggcccttccgtgttcctgttccccccaaagcccaaggacaccctgatgatctcccggacccccgaagtgacctgcgtggtggtggatgtgtcccacgaggaccccgaggtgcagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagttcaactccaccttccgggtggtgtccgtgctgaccgtggtgcaccaggactggctgaacggcaaagagtacaagtgcaaggtgtccaacaagggcctgcctgcccccatcgaaaagaccatctccaagacaaagggccagccccgcgagcctcaggtgtacacactgcctcccagccgggaagagatgaccaagaaccaggtgtccctgacctgtctggtcaagggcttctacccctccgatatcgccgtggaatgggagtccaacggccagcccgagaacaactacaagaccaccccccccatgctggactccgacggctcattcttcctgtactccaagctgacagtggacaagtcccggtggcagcagggcaacgtgttctcctgctctgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagccccggcaaa 222 mAb-CD73.6-Vh-caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccct IhHC-IgG2-C219S-gagactctcctgtgcagcctctggattcaccttcagtaactatggcatgcactggg gG1.1ftccgccaggctccaggcaaggggctggagtgggtggcagttatattgtatgattccagtaataaatactatccagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagagggggcagcagctggtaccctgattcttttgatatctggggccaaggaacaatggtcaccgtctcttcagcgtcgaccaagggcccatcggtcttccccctggcgccctgctccaggagcacctccgagagcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacaccttcccagctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcaacttcggcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagttgagcgcaaatcctgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccaagcagcatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaa 223 mAb-CD73.7-Vh-caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccct hHC-IgG1.1fgagactctcctgtgcaagctctggattcaccttcagtaactatggcatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatattgtatgattccagtaataaatactatccagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagagggggcagcagctggtaccctgattcttttgatatctggggccaaggaacaatggtcaccgtctcttcagcgtcgaccaagggcccctccgtgtttcctctggccccttccagcaagtccacctctggcggaacagccgctctgggctgcctggtcaaggactacttccccgagcctgtgaccgtgtcctggaactctggcgccctgacatctggcgtgcacaccttccctgctgtgctgcagtctagcggcctgtactccctgtcctccgtcgtgacagtgccctccagctctctgggcacccagacctacatctgcaacgtgaaccacaagccctccaacaccaaggtggacaagcgggtggaacccaagtcctgcgacaagacccatacctgccctccctgccctgctcctgaagctgaaggcgcccctagcgtgttcctgttccctccaaagcccaaggacaccctgatgatctcccggacccctgaagtgacctgcgtggtggtggatgtgtcccacgaggacccagaagtgaagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcctagagaggaacagtacaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggattggctgaacggcaaagagtacaagtgcaaggtgtccaacaaggccctgcctagctccatcgaaaagaccatctccaaggctaagggccagccccgcgagccccaggtgtacacactgcctccatcccgggaagagatgaccaagaaccaggtgtccctgacttgcctcgtgaagggcttctacccctccgatatcgccgtggaatgggagtccaacggccagcctgagaacaactacaagaccacccctcccgtgctggactccgacggctcattcttcctgtacagcaagctgacagtggataagtcccggtggcagcaggggaacgtgttctcctgctccgtgatgcacgaggctctgcacaaccactacacacagaagtccctgtctctgtcccctggc 224 mAb-CD73.7-Vh-caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccct hHC-IgG2-C219Sgagactctcctgtgcaagctctggattcaccttcagtaactatggcatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatattgtatgattccagtaataaatactatccagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagagggggcagcagctggtaccctgattcttttgatatctggggccaaggaacaatggtcaccgtctcttcagcgtcgaccaagggcccctctgtgtttcctctggccccttgctcccggtccacctctgagtctaccgctgctctgggctgcctggtcaaggactacttccccgagcctgtgaccgtgtcctggaactctggcgctctgacctccggcgtgcacacctttccagccgtgctgcagtcctccggcctgtactctctgtcctccgtcgtgaccgtgccctcctccaacttcggcacccagacctacacctgtaacgtggaccacaagccctccaacaccaaggtggacaagaccgtggaacggaagtcctgcgtggaatgccctccttgccctgcacctcctgtggctggcccttccgtgttcctgttccccccaaagcccaaggacaccctgatgatctcccggacccccgaagtgacctgcgtggtggtggatgtgtcccacgaggaccccgaggtgcagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagttcaactccaccttccgggtggtgtccgtgctgaccgtggtgcaccaggactggctgaacggcaaagagtacaagtgcaaggtgtccaacaagggcctgcctgcccccatcgaaaagaccatctccaagacaaagggccagccccgcgagcctcaggtgtacacactgcctcccagccgggaagagatgaccaagaaccaggtgtccctgacctgtctggtcaagggcttctacccctccgatatcgccgtggaatgggagtccaacggccagcccgagaacaactacaagaccaccccccccatgctggactccgacggctcattcttcctgtactccaagctgacagtggacaagtcccggtggcagcagggcaacgtgttctcctgctctgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagccccggcaaa 225 mAb-CD73.7-Vh-caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccct hHC-IgG2-C219S-gagactctcctgtgcaagctctggattcaccttcagtaactatggcatgcactggg IgG1.1ftccgccaggctccaggcaaggggctggagtgggtggcagttatattgtatgattccagtaataaatactatccagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagagggggcagcagctggtaccctgattcttttgatatctggggccaaggaacaatggtcaccgtctcttcagcgtcgaccaagggcccatcggtcttccccctggcgccctgctccaggagcacctccgagagcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacaccttcccagctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcaacttcggcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagttgagcgcaaatcctgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccaagcagcatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaa 226 mAb-CD73.8-Vh-caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccct hHC-IgG1.1fgagactctcctgtgcagcctctggattcaccttcagtaactatggcatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatatggtatgattccagtaataaatactatccagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagagggggcagcagctggtaccctgattcttttgatatctggggccaaggaacaatggtcaccgtctcttcagcgtcgaccaagggcccctccgtgtttcctctggccccttccagcaagtccacctctggcggaacagccgctctgggctgcctggtcaaggactacttccccgagcctgtgaccgtgtcctggaactctggcgccctgacatctggcgtgcacaccttccctgctgtgctgcagtctagcggcctgtactccctgtcctccgtcgtgacagtgccctccagctctctgggcacccagacctacatctgcaacgtgaaccacaagccctccaacaccaaggtggacaagcgggtggaacccaagtcctgcgacaagacccatacctgccctccctgccctgctcctgaagctgaaggcgcccctagcgtgttcctgttccctccaaagcccaaggacaccctgatgatctcccggacccctgaagtgacctgcgtggtggtggatgtgtcccacgaggacccagaagtgaagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcctagagaggaacagtacaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggattggctgaacggcaaagagtacaagtgcaaggtgtccaacaaggccctgcctagctccatcgaaaagaccatctccaaggctaagggccagccccgcgagccccaggtgtacacactgcctccatcccgggaagagatgaccaagaaccaggtgtccctgacttgcctcgtgaagggcttctacccctccgatatcgccgtggaatgggagtccaacggccagcctgagaacaactacaagaccacccctcccgtgctggactccgacggctcattcttcctgtacagcaagctgacagtggataagtcccggtggcagcaggggaacgtgttctcctgctccgtgatgcacgaggctctgcacaaccactacacacagaagtccctgtctctgtcccctggc 227 mAb-CD73.8-Vh-caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccct hHC-IgG2-C219Sgagactctcctgtgcagcctctggattcaccttcagtaactatggcatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatatggtatgattccagtaataaatactatccagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagagggggcagcagctggtaccctgattcttttgatatctggggccaaggaacaatggtcaccgtctcttcagcgtcgaccaagggcccctctgtgtttcctctggccccttgctcccggtccacctctgagtctaccgctgctctgggctgcctggtcaaggactacttccccgagcctgtgaccgtgtcctggaactctggcgctctgacctccggcgtgcacacctttccagccgtgctgcagtcctccggcctgtactctctgtcctccgtcgtgaccgtgccctcctccaacttcggcacccagacctacacctgtaacgtggaccacaagccctccaacaccaaggtggacaagaccgtggaacggaagtcctgcgtggaatgccctccttgccctgcacctcctgtggctggcccttccgtgttcctgttccccccaaagcccaaggacaccctgatgatctcccggacccccgaagtgacctgcgtggtggtggatgtgtcccacgaggaccccgaggtgcagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagttcaactccaccttccgggtggtgtccgtgctgaccgtggtgcaccaggactggctgaacggcaaagagtacaagtgcaaggtgtccaacaagggcctgcctgcccccatcgaaaagaccatctccaagacaaagggccagccccgcgagcctcaggtgtacacactgcctcccagccgggaagagatgaccaagaaccaggtgtccctgacctgtctggtcaagggcttctacccctccgatatcgccgtggaatgggagtccaacggccagcccgagaacaactacaagaccaccccccccatgctggactccgacggctcattcttcctgtactccaagctgacagtggacaagtcccggtggcagcagggcaacgtgttctcctgctctgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagccccggcaaa 228 mAb-CD73.8-Vh-caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccct hHC-IgG2-C219S-gagactctcctgtgcagcctctggattcaccttcagtaactatggcatgcactggg IgG1.1ftccgccaggctccaggcaaggggctggagtgggtggcagttatatggtatgattccagtaataaatactatccagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagagggggcagcagctggtaccctgattcttttgatatctggggccaaggaacaatggtcaccgtctcttcagcgtcgaccaagggcccatcggtcttccccctggcgccctgctccaggagcacctccgagagcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacaccttcccagctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcaacttcggcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagttgagcgcaaatcctgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccaagcagcatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaa 229 mAb-CD73.9-Vh-caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccct hHC-IgG1.1fgagactctcctgtgcaagctctggattcaccttcagtaactatggcatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatatggtatgattccagtaataaatactatccagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagagggggcagcagctggtaccctgattcttttgatatctggggccaaggaacaatggtcaccgtctcttcagcgtcgaccaagggcccctccgtgtttcctctggccccttccagcaagtccacctctggcggaacagccgctctgggctgcctggtcaaggactacttccccgagcctgtgaccgtgtcctggaactctggcgccctgacatctggcgtgcacaccttccctgctgtgctgcagtctagcggcctgtactccctgtcctccgtcgtgacagtgccctccagctctctgggcacccagacctacatctgcaacgtgaaccacaagccctccaacaccaaggtggacaagcgggtggaacccaagtcctgcgacaagacccatacctgccctccctgccctgctcctgaagctgaaggcgcccctagcgtgttcctgttccctccaaagcccaaggacaccctgatgatctcccggacccctgaagtgacctgcgtggtggtggatgtgtcccacgaggacccagaagtgaagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcctagagaggaacagtacaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggattggctgaacggcaaagagtacaagtgcaaggtgtccaacaaggccctgcctagctccatcgaaaagaccatctccaaggctaagggccagccccgcgagccccaggtgtacacactgcctccatcccgggaagagatgaccaagaaccaggtgtccctgacttgcctcgtgaagggcttctacccctccgatatcgccgtggaatgggagtccaacggccagcctgagaacaactacaagaccacccctcccgtgctggactccgacggctcattcttcctgtacagcaagctgacagtggataagtcccggtggcagcaggggaacgtgttctcctgctccgtgatgcacgaggctctgcacaaccactacacacagaagtccctgtctctgtcccctggc 230 mAb-CD73.9-Vh-caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccct hHC-IgG2-C219Sgagactctcctgtgcaagctctggattcaccttcagtaactatggcatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatatggtatgattccagtaataaatactatccagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagagggggcagcagctggtaccctgattcttttgatatctggggccaaggaacaatggtcaccgtctcttcagcgtcgaccaagggcccctctgtgtttcctctggccccttgctcccggtccacctctgagtctaccgctgctctgggctgcctggtcaaggactacttccccgagcctgtgaccgtgtcctggaactctggcgctctgacctccggcgtgcacacctttccagccgtgctgcagtcctccggcctgtactctctgtcctccgtcgtgaccgtgccctcctccaacttcggcacccagacctacacctgtaacgtggaccacaagccctccaacaccaaggtggacaagaccgtggaacggaagtcctgcgtggaatgccctccttgccctgcacctcctgtggctggcccttccgtgttcctgttccccccaaagcccaaggacaccctgatgatctcccggacccccgaagtgacctgcgtggtggtggatgtgtcccacgaggaccccgaggtgcagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagttcaactccaccttccgggtggtgtccgtgctgaccgtggtgcaccaggactggctgaacggcaaagagtacaagtgcaaggtgtccaacaagggcctgcctgcccccatcgaaaagaccatctccaagacaaagggccagccccgcgagcctcaggtgtacacactgcctcccagccgggaagagatgaccaagaaccaggtgtccctgacctgtctggtcaagggcttctacccctccgatatcgccgtggaatgggagtccaacggccagcccgagaacaactacaagaccaccccccccatgctggactccgacggctcattcttcctgtactccaagctgacagtggacaagtcccggtggcagcagggcaacgtgttctcctgctctgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagccccggcaaa 231 mAb-CD73.9-Vh-caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccct hHC-IgG2-C219S-gagactctcctgtgcaagctctggattcaccttcagtaactatggcatgcactggg IgG1.1ftccgccaggctccaggcaaggggctggagtgggtggcagttatatggtatgattccagtaataaatactatccagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagagggggcagcagctggtaccctgattcttttgatatctggggccaaggaacaatggtcaccgtctcttcagcgtcgaccaagggcccatcggtcttccccctggcgccctgctccaggagcacctccgagagcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacaccttcccagctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcaacttcggcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagttgagcgcaaatcctgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccaagcagcatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaa 232 mAb-CD73.10-Vh-caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccct hHC-IgG1.1fgagactctcctgtgcagcctctggattcaccttcagtaactatggcatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatatggtatgatgagagtaataaatactatccagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagagggggcagcagctggtaccctgattcttttgatatctggggccaaggaacaatggtcaccgtctcttcagcgtcgaccaagggcccctccgtgtttcctctggccccttccagcaagtccacctctggcggaacagccgctctgggctgcctggtcaaggactacttccccgagcctgtgaccgtgtcctggaactctggcgccctgacatctggcgtgcacaccttccctgctgtgctgcagtctagcggcctgtactccctgtcctccgtcgtgacagtgccctccagctctctgggcacccagacctacatctgcaacgtgaaccacaagccctccaacaccaaggtggacaagcgggtggaacccaagtcctgcgacaagacccatacctgccctccctgccctgctcctgaagctgaaggcgcccctagcgtgttcctgttccctccaaagcccaaggacaccctgatgatctcccggacccctgaagtgacctgcgtggtggtggatgtgtcccacgaggacccagaagtgaagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcctagagaggaacagtacaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggattggctgaacggcaaagagtacaagtgcaaggtgtccaacaaggccctgcctagctccatcgaaaagaccatctccaaggctaagggccagccccgcgagccccaggtgtacacactgcctccatcccgggaagagatgaccaagaaccaggtgtccctgacttgcctcgtgaagggcttctacccctccgatatcgccgtggaatgggagtccaacggccagcctgagaacaactacaagaccacccctcccgtgctggactccgacggctcattcttcctgtacagcaagctgacagtggataagtcccggtggcagcaggggaacgtgttctcctgctccgtgatgcacgaggctctgcacaaccactacacacagaagtccctgtctctgtcccctggc 233 mAb-CD73.10-Vh-caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccct hHC-IgG2-C219Sgagactctcctgtgcagcctctggattcaccttcagtaactatggcatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatatggtatgatgagagtaataaatactatccagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagagggggcagcagctggtaccctgattcttttgatatctggggccaaggaacaatggtcaccgtctcttcagcgtcgaccaagggcccctctgtgtttcctctggccccttgctcccggtccacctctgagtctaccgctgctctgggctgcctggtcaaggactacttccccgagcctgtgaccgtgtcctggaactctggcgctctgacctccggcgtgcacacattccagccgtgctgcagtcctccggcctgtactctctgtcctccgtcgtgaccgtgccctcctccaacttcggcacccagacctacacctgtaacgtggaccacaagccctccaacaccaaggtggacaagaccgtggaacggaagtcctgcgtggaatgccctccttgccctgcacctcctgtggctggcccttccgtgttcctgttccccccaaagcccaaggacaccctgatgatctcccggacccccgaagtgacctgcgtggtggtggatgtgtcccacgaggaccccgaggtgcagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagttcaactccaccttccgggtggtgtccgtgctgaccgtggtgcaccaggactggctgaacggcaaagagtacaagtgcaaggtgtccaacaagggcctgcctgcccccatcgaaaagaccatctccaagacaaagggccagccccgcgagcctcaggtgtacacactgcctcccagccgggaagagatgaccaagaaccaggtgtccctgacctgtctggtcaagggcttctacccctccgatatcgccgtggaatgggagtccaacggccagcccgagaacaactacaagaccaccccccccatgctggactccgacggctcattcttcctgtactccaagctgacagtggacaagtcccggtggcagcagggcaacgtgttctcctgctctgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagccccggcaaa 234 mAb-CD73.10-Vh-caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccct hHC-IgG2-C219S-gagactctcctgtgcagcctctggattcaccttcagtaactatggcatgcactggg IgG1.1ftccgccaggctccaggcaaggggctggagtgggtggcagttatatggtatgatgagagtaataaatactatccagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagagggggcagcagctggtaccctgattcttttgatatctggggccaaggaacaatggtcaccgtctcttcagcgtcgaccaagggcccatcggtcttccccctggcgccctgctccaggagcacctccgagagcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacaccttcccagctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcaacttcggcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagttgagcgcaaatcctgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccaagcagcatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaa 235 mAb-CD73.11-Vh-caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccct hHC-IgG1.1fgagactctcctgtgcagcgtctggattcaccttcagtaactatggcatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatatggtatgatgaaagtaataaatactatgcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtttctgcaaatgaacagcctgagagccgaggacacggctgtgtattattgtgcgagagggtataacagcaggtggtaccctgatgcttttgatatctggggccaagggacaatggtcaccgtctcttcagcgtcgaccaagggcccctccgtgtttcctctggccccttccagcaagtccacctctggcggaacagccgctctgggctgcctggtcaaggactacttccccgagcctgtgaccgtgtcctggaactctggcgccctgacatctggcgtgcacaccttccctgctgtgctgcagtctagcggcctgtactccctgtcctccgtcgtgacagtgccctccagctctctgggcacccagacctacatctgcaacgtgaaccacaagccctccaacaccaaggtggacaagcgggtggaacccaagtcctgcgacaagacccatacctgccctccctgccctgctcctgaagctgaaggcgcccctagcgtgttcctgttccctccaaagcccaaggacaccctgatgatctcccggacccctgaagtgacctgcgtggtggtggatgtgtcccacgaggacccagaagtgaagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcctagagaggaacagtacaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggattggctgaacggcaaagagtacaagtgcaaggtgtccaacaaggccctgcctagctccatcgaaaagaccatctccaaggctaagggccagccccgcgagccccaggtgtacacactgcctccatcccgggaagagatgaccaagaaccaggtgtccctgacttgcctcgtgaagggcttctacccctccgatatcgccgtggaatgggagtccaacggccagcctgagaacaactacaagaccacccctcccgtgctggactccgacggctcattcttcctgtacagcaagctgacagtggataagtcccggtggcagcaggggaacgtgttctcctgctccgtgatgcacgaggctctgcacaaccactacacacagaagtccctgtctctgtcccctgg c 236mAb-CD73.11-Vh- caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtcccthHC-IgG2-C219S gagactctcctgtgcagcgtctggattcaccttcagtaactatggcatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatatggtatgatgaaagtaataaatactatgcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtttctgcaaatgaacagcctgagagccgaggacacggctgtgtattattgtgcgagagggtataacagcaggtggtaccctgatgcttttgatatctggggccaagggacaatggtcaccgtctcttcagcgtcgaccaagggcccctctgtgtttcctctggccccttgctcccggtccacctctgagtctaccgctgctctgggctgcctggtcaaggactacttccccgagcctgtgaccgtgtcctggaactctggcgctctgacctccggcgtgcacacctttccagccgtgctgcagtcctccggcctgtactctctgtcctccgtcgtgaccgtgccctcctccaacttcggcacccagacctacacctgtaacgtggaccacaagccctccaacaccaaggtggacaagaccgtggaacggaagtcctgcgtggaatgccctccttgccctgcacctcctgtggctggcccttccgtgttcctgttccccccaaagcccaaggacaccctgatgatctcccggacccccgaagtgacctgcgtggtggtggatgtgtcccacgaggaccccgaggtgcagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagttcaactccaccttccgggtggtgtccgtgctgaccgtggtgcaccaggactggctgaacggcaaagagtacaagtgcaaggtgtccaacaagggcctgcctgcccccatcgaaaagaccatctccaagacaaagggccagccccgcgagcctcaggtgtacacactgcctcccagccgggaagagatgaccaagaaccaggtgtccctgacctgtctggtcaagggcttctacccctccgatatcgccgtggaatgggagtccaacggccagcccgagaacaactacaagaccaccccccccatgctggactccgacggctcattcttcctgtactccaagctgacagtggacaagtcccggtggcagcagggcaacgtgttctcctgctctgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagccccggcaaa 237 CD73.4 (VH) - caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccct Nucleotide gagactctcctgtgcagcctctggattcaccttcagtaactatggcatgcactggg sequencetccgccaggctccaggcaaggggctggagtgggtggcagttatattgtatgatggaagtaataaatactatccagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagagggggcagcagctggtaccctgattcttttgatatctggggccaaggaacaatggtcaccgtctcttca 238 5F8 VK3EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFT LTISSLEPEDFAVYYCQQRSNWWTFGQGTKVEIK239 5F8 VK3 CDR1 RASQSVSSYLA 240 5F8 VK3 CDR2 DASNRAT 241 5F8 VK3 CDR3QQRSNWWT 242 5F8 VK3 -  GAGGTGCAGCTGGTGGAGTCCGGGGGAGGCTTA Nucleotide GTTCAGCCTGGGGGGTCCCTGAGACTCTCCTGTG sequenceCAGCCTCTGGATTCACCTTCAGTAGCTACTGGAT GCACTGGGTCCGCCAAGCTCCAGGGAAGGGGCTGGTGTGGGTCTCACGTATTATTAGTGATGGGAGT AGCACAGGTTACGCGGATTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACG CTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCAAGAGAGTTTA GCAGTGGCTGGTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA 243 11F11 (full lengthCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTG heavy chain) - GTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTG NT SeqCAACGTCTGGATTCACCTTCAGTAACTATGGCAT GCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATTGTATGATGGAAGT AATAAATACTATCCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGC TGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGGCA GCAGCTGGTACCCTGATTCTTTTGATATCTGGGGCCAAGGAACAATGGTCACCGTCTCTTCAGCCTCC ACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCC TGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACC AGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGAC CGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACC AAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGG CAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAG GTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGAC GGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTG GTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCA ACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCAC AGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGT CAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTA CAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACA AGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTA CACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA244 11F11 (full length GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTlight chain 1) -  CTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTG NT SeqCAGGGCCAGTCAGGGTGTTAGCAGCTACTTAGCC TGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTG GCATCCCAGCCAGGTTCAGTGGCAGTGGGCCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGA GCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCATCTCACTTTCGGCGGAGGGA CCAAGGTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCA GTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTAC AGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCA AGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAG TCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGA GTGT 245 11F11 (full lengthGACATCCAGATGACCCAGTCTCCATCCTCACTGT light chain 2) - CTGCATCTGTAGGAGACAGAGTCACCATCACTTG NT SeqTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCC TGGTATCAGCAGAAACCAGAGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTTTGCAAAGTG GGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCA GCCTGAAGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTACCCTCTCACTTTCGGCGGAGGGA CCAAGGTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCA GTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTAC AGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCA AGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAG TCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGA GTGT 246 4C3 (full lengthGAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTG heavy chain) - GTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTG NT SeqCAGCCTCTGGATTCACCTTTGATGATTATGCCAT GCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAAGAGTGG TAGCATAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCC CTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGTAAAAGGGTATT ACGTTATTTTGACTGGCCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCT GTCTCCGGGTAAA 2474C3 (full length GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGT light chain 1) - CTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTG NT SeqCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCC TGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTG GCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGA GCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCGCTCACTTTCGGCGGAGGGA CCAAGGTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCA GTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTAC AGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCA AGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAG TCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGA GTGT 248 4C3 (full lengthGACATCCAGATGACCCAGTCTCCATCCTCACTGT light chain 2) - CTGCATCTGTAGGAGACAGAGTCACCTTCACTTG NT SeqTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCC TGGTATCAGCAGAAACCAGAGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTTTGCAAAGTG GGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCA GCCTGAAGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTACCCTCCAACGTTCGGCCAGGGGA CCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCA GTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTAC AGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCA AGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAG TCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGA GTGT 249 4C3 (full lengthGACATCCAGATGACCCAGTCTCCATCCTCACTGT light chain 3) - CTGCATCTGTAGGAGACAGAGTCACCTTCACTTG NT SeqTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCC TGGTATCAGCAGAAACCAGAGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTTTGCAAAGTG GGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCA GCCTGAAGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTACCCTCCAACGTTCGGCCAAGGGA CCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCA GTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTAC AGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCA AGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAG TCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGA GTGT 250 4D4 (full lengthCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTG heavy chain) - GTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTG NT SeqCAGCGTCTGGATTCACCTTCAGTAACTATGGCAT GCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGTATGATGAAAG TAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG CTGTTTCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTATTGTGCGAGAGGGTATA ACAGCAGGTGGTACCCTGATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGCC TCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGG CCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTG ACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT GACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAAC ACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTG TGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCT GAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTG GACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGT GTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCT CCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAAC CACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCC TGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACA ACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTG GACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACC ACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 251 4D4 (full length GACATCCAGATGACCCAGTCTCCATCCTCACTGTlight chain 1) -  CTGCATCTGTAGGAGACAGAGTCACCATCACTTG NT SeqTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCC TGGTATCAGCAGAAACCAGAGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTTTGCAAAGTG GGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCA GCCTGAAGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTACCCGCTCACTTTCGGCGGAGGGA CCAAGGTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCA GTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTAC AGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCA AGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAG TCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGA GTGT 252 10D2 (full lengthCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTG heavy chain) - GTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTG NT SeqCAGCGTCTGGATTCACCTTCAGTAACTATGGCCT GCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATACGGTATGATGGAAG TAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG CTGTATCTGCAAATGAGCAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGGGGGGGC AGCAGCTGGTACCCGGACGGTTTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTT CCACCAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGC CCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTG ACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT GACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAA CACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCATCATGCCCAGCACCTGAG TTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGAC CCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTA CGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTA CCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAG GTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAG AGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTG CCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAAC AACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGT GGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAAC CACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAA 253 10D2 (full length GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTlight chain 1) -  CTGCATCTGTAGGAGACAGAGTCACCATCACTTG NT SeqCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCC TGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTG GGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCA GCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCCACTTTCGGCGGAGGGACCA AGGTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTT GAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAG TGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAG GACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTC TACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGT GT 254 10D2 (full lengthGACATCCAGATGACCCAGTCTCCATCCTCACTGT light chain 2) - CTGCATCTGTAGGAGACAGAGTCACCATCACTTG NT SeqTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCC TGGTATCAGCAGAAACCAGAGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTTTGCAAAGTG GGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCA GCCTGAAGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTACCCGCTCACTTTCGGCGGAGGGA CCAAGGTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCA GTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTAC AGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCA AGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAG TCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGA GTGT 255 11A6 (full lengthGAAGTGCAGCTGGTGGAATCTGGGGGAAACTTG heavy chain) - GTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTG NT SeqCAGCCTCTGGATTCACCTTTGATGATTATGCCAT GCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATAATAAT GACATAGGCTATGCGGACTCTGTGAAGGGCCGATTCATCATCTCCAGAGACAACGCCAAGAACTCCC TGTATCTGCAAATGAACAGTCTGAGACCTGAGGACACGGCCTTGTATTATTGTGTAAAAGGTTATTA CGTTATTTTGACTGGTCTTGACTACTGGGGCCAGGGAACCCCGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCT GTCTCCGGGTAAA 25611A6 (full length GACATCCAGATGACCCAGTCTCCATCCTCACTGT light chain 1) - CTGCATCTGTAGGAGACAGAGTCACCATCACTTG NT SeqTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCC TGGTATCAGCAGAAACCAGAGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTTTGCAAAGTG GGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCA GCCTGAAGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTACCCGCTCACTTTCGGCGGAGGGA CCAAGGTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCA GTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTAC AGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCA AGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAG TCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGA GTGT 257 24H2 (full lengthCAGGTGCAACTGGTGGAGTCTGGGGGAGGCGTG heavy chain) - GTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTG NT SeqCAGCGTCTGGATTCACCTTCAGTAACTATGGCAT GCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGTATGATGGAGG TAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG CTGTTTCTGCAAATGAACAGCCTGAGAGCCGAAGACACGGCTGTGTATTACTGTGCGAGAGGGGGC AGCAGCTGGTACCCTGATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGCTTC CACCAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGA CCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTG ACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACA CCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCATCATGCCCAGCACCTGAGTT CCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCC CTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACG TGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACC GTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGT CTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAG CCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCC TGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACA ACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTG GACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACC ACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAA 258 24H2 (full length GACATCCAGATGACCCAGTCTCCATCCTCACTGTlight chain 1) -  CTGCATCTGTAGGAGACAGAGTCACCATCACTTG NT SeqTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCC TGGTATCAGCAGAAACCAGAGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTTTGCAAAGTG GGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCA GCCTGAAGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTACCCTCTCACTTTCGGCGGAGGGA CCAAGGTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCA GTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTAC AGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCA AGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAG TCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGA GTGT 259 5F8 (full lengthGAGGTGCAGCTGGTGGAGTCCGGGGGAGGCTTA heavy chain) - GTTCAGCCTGGGGGGTCCCTGAGACTCTCCTGTG NT SeqCAGCCTCTGGATTCACCTTCAGTAGCTACTGGAT GCACTGGGTCCGCCAAGCTCCAGGGAAGGGGCTGGTGTGGGTCTCACGTATTATTAGTGATGGGAGT AGCACAGGTTACGCGGATTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACG CTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCAAGAGAGTTTA GCAGTGGCTGGTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCT CCGGGTAAA 2605F8 (full length GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGT light chain 1) - CTGCATCTGTAGGAGACAGAGTCACCATCACTTG NT SeqCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCC TGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTG GGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCA GCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAGTAGTTACCCTCGGACGTTCGGCCAAGGGA CCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCA GTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTAC AGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCA AGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAG TCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGA GTGT 261 5F8 (full lengthGACATCCAGATGACCCAGTCTCCATCCTCACTGT light chain 2) - CTGCATCTGTAGGAGACAGAGTCACCATCACTTG NT SeqTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCC TGGTATCAGCAGAAACCAGAGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTTTGCAAAGTG GGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGGTTTCACTCTCACCATCAGCAGCCTGCA GCCTGAAGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTACCCTCGGACGTTCGGCCAAGGGA CCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCA GTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTAC AGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCA AGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAG TCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGA GTGT 262 6E11 (full lengthGAAGTGCAGCTGGTGGAGTCTGGGGGAGCCTTG heavy chain) - GTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTG NT SeqCAGCCTCTGGATTCACCTTTGATGATTATGCCAT GCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTACTTGGAATAGTGGT GGCATAGGCTACGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCC TGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATAGGTA TTACAGCAGTTGGCTCCTCTTTGACAACTGGGGCCAGGGAATTCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCC CTGTCTCCGGGTAAA 2636E11 (full length GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGT light chain 1) - CTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTG NT SeqCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTT AGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCC ACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGAC TGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCATTATGGTAGCTCATTCACTTTCGGCCCTGGG ACCAAAGTGGATATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGC AGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGT ACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAG CAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAA AGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGA GAGTGT 264 7A11 (full lengthGAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTG heavy chain) - GTACAGACTGGCAGGTCCCTGAGACTCTCCTGTG NT SeqCAGCCTCTGGATTCACCTTTGATGATTATGCCAT GCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGATATTAGTTGGAATAGTGAT ATTATAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCTAGAGACAACGCCAAGAACTCCCT GTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATATTTAT GGTTCGGGGAGTTCTTTTTTTGACTACTGGGGCCAGGGAATCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCC CTGTCTCCGGGTAAA 2657A11 (full length GACATCCAGATGACCCAGTCTCCATCCTCACTGT light chain 1) -CTGCATCTGTAGGAGACAGAGTCACCATCACTTG NT SeqTCGGGCGAGTCAGTATATTAGCAGCTGGTTAGCC   TGGTATCAGCAGAAACCAGAGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTTTGCAAAGTG GGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCA GCCTGAAGATTTTGCAACTTATTACTGCCAACAGTATCATAGTTACCCTCCCACCTTCGGCCAAGGGA CACGACTGGAGATTAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCA GTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTAC AGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCA AGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAG TCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGA GTGT 266 CD73.4.IgG2C219caggtgcagc tggtggagtc tgggggaggc SIgG1.1f -gtggtccagc ctgggaggtc cctgagactc Alternativetcctgtgcag cctctggatt caccttcagt NT Seq aactatggca tgcactgggt ccgccaggctccaggcaagg ggctggagtg ggtggcagtt atattgtatg atggaagtaa taaatactatccagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtatctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gagagggggcagcagctggt accctgattc ttttgatatc tggggccaag gaacaatggt caccgtctcttcagcgtcga ccaagggccc atcggtcttc cccctggcgc cctgctccag gagcacctccgagagcacag cggccctggg ctgcctggtc aaggactact tccccgaacc ggtgacggtgtcgtggaact caggcgctct gaccagcggc gtgcacacct tcccagctgt cctacagtcctcaggactct actccctcag cagcgtggtg accgtgccct ccagcaactt cggcacccagacctacacct gcaacgtaga tcacaagccc agcaacacca aggtggacaa gacagttgagcgcaaatcct gtgtcgagtg cccaccgtgc ccagcaccac ctgtggcagg accgtcagtcttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcacatgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggacggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtaccgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaagtgcaaggtct ccaacaaagc cctcccaagc agcatcgaga aaaccatctc caaagccaaagggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaagaaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggagtgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactccgacggctcct tcttcctcta tagcaagctc accgtggaca agagcaggtg gcagcaggggaacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagcctctccctgt ccccgggttg a 267 IgG1f ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK 268 IgG2.3ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK 269 IgG2.3G1-AYASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 270 IgG2.3G1-KHASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK 271 IgG2.5ASTKGPSVFPLAPSSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK 272 IgG1.1fASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK 273 IgG2.3G1.1f-KHASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK 274 IgG1-deltaTHTASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 275 IgG2.3-plusTHTASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVETHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFELYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 276 IgG2.3-plusGGGASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVEGGGCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFELYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 277 IgG2.5G1.1f-KHASTKGPSVFPLAPSSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK 278 IgG2.5G1-AYASTKGPSVFPLAPSSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 279 IgG2.5G1-KHASTKGPSVFPLAPSSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK 280 IgG2.5-plusTHTASTKGPSVFPLAPSSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVETHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 281 IgG1-G2.3G1-AYASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVERKSCVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 282 IgG1-G2.3G1-KHASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK 283 CD73 from FIG. XCPRAARAPATLLLALGAVLWPAAGAWELTILHTND 27AVHSRLEQTSEDSSKCVNASRCMGGVARLFTKVQQIRRAEPNVLLLDAGDQYQGTIWFTVYKGAEVAHFMNALRYDAMALGNHEFDNGVEGLIEPLLKEAKFPILSANIKAKGPLASQISGLYLPYKVLPVGDEVVGIVGYTSKETPFLSNPGTNLVFEDEITALQPEVDKLKTLNVNKIIALGHSGFEMDKLIAQKVRGVDVVVGGHSNTFLYTGNPPSKEVPAGKYPFIVTSDDGRKVPVVQAYAFGKYLGYLKIEFDERGNVISSHGNPILLNSSIPEDPSIKADINKWRIKLDNYSTQELGKTIVYLDGSSQSCRFRECNMGNLICDAMINNNLRHADETFWNHVSMCILNGGGIRSPIDERNNGTITWENLAAVLPFGGTFDLVQLKGSTLKKAFEHSVHRYGQSTGEFLQVGGIHVVYDLSRKPGDRVVKLDVLCTKCRVPSYDPLKMDEVYKVILPNFLANGGDGFQMIKDELLRHDSGDQDINVVSTYISKMKVIYP AVEGRIKHHHHHH 284 Hinge region VDKRV amino acid 285 Hinge region  VDKTV amino acid 286 Hinge region EPKSCDKTHT amino acid 287 Hinge region  ELKTPLGDTTHT amino acid 288Hinge region  EPKS amino acid 289 Hinge region  ESKYGPP amino acid 290Hinge region  CPPCP amino acid 291 Hinge region  CCVECPPCP amino acid292 Hinge region  CPRCP (EPKSCDTPPPCPRCP)₃ amino acid 293 Hinge region CPRCP (EPKSCDTPPPCPRCP)₂ amino acid 294 Hinge region CPRCP (EPKSCDTPPPCPRCP)₁ amino acid 295 Hinge region CDTPPPCPRCP (EPKSCDTPPPCPRCP)₂ amino acid 296 Hinge region  CDTPPPCPRCPamino acid 297 Hinge region  CPSCP amino acid 298 Hinge region  APELLGGamino acid 299 Hinge region  APPVAG amino acid 300 G2-G1-G1-G1ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 301 G2.5-G1-G1-G1ASTKGPSVFPLAPSRSTSESTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 302 G1-G2.3-G2-G2ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCVE CPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK 303 G1-KRGEGSSNLFASTKGPSVFPLAPSSRSTSESTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 304 G1-KRGEGSASTKGPSVFPLAPSSRSTSESTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEAL HNHYTQKSLSLSPGK 305 G1-SNLFASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNEGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRIPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEAL HNHYTQKSLSLSPGK 306 IgG1-ITNDRTPRASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVDHKPSNTKVDKTVERKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRIPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEAL HNHYTQKSLSLSPGK 307 G1-SNLFPRASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNEGTQTYICNVNHKPSNTKVDKRVERKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRIPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEAL HNHYTQKSLSLSPG 308 G2-RKEGSGNSFLASTKGPSVFPLAPCSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVDHKPSNTKVDKTVERKSCVE CPPCPAPPVAGPSVFLFPPKPKDTLMISRIPEVICVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK 309 G2-RKEGSGASTKGPSVFPLAPCSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNEGTQTYTCNVDHKPSNTKVDKTVERKSCVE CPPCPAPPVAGPSVFLFPPKPKDTLMISRIPEVICVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK 310 G2-NSFLASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVDHKPSNTKVDKTVERKSCVE CPPCPAPPVAGPSVFLFPPKPKDTLMISRIPEVICVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK 311 IgG2-TIDNTRRPASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNEGTQTYICNVNHKPSNTKVDKRVEPKSCVE CPPCPAPPVAGPSVFLFPPKPKDTLMISRIPEVICVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK 312 G2-NSFLRPASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVDHKPSNTKVDKTVEPKSCVE CPPCPAPPVAGPSVFLFPPKPKDTLMISRIPEVICVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK 313 G1-G1-G2-G1-AYASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRIPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPR EEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEAL HNHYTQKSLSLSPGK 314 G1-G1-G2-G1-KHASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPRE EQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK 315 G2-G2.3-G1-G2-KHASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVE CPPCPAPPVAGPSVFLFPPKPKDTLMISRIPEVICVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK 316 G2.5-G2.3-G1-G2-KHASTKGPSVFPLAPSSRSTSESTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVE CPPCPAPPVAGPSVFLFPPKPKDTLMISRIPEVICVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK 317 G2-G2.3-G1-G2-AYASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVE CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG 318 G2.5-G2.3-G1-G2-AYASTKGPSVFPLAPSRSTSESTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVE CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK 319 G1-G2.3-G1-G1-KHASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCVE CPPCPAPPVAGPSVFLFPPKPKDTLMISRIPEVICVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK 320 G2-G1-G2-G2-AYASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRIPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPR EEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 321 G2.5-G1-G2-G2-AYASTKGPSVFPLAPSRSTSESTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRIPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPR EEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 322 G1-G2-G1-G1-AYASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCVE CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK 323 G2-G1-G2-G2-KHASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCDK THTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPRE EQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG 324 G2.5-G1-G2-G2-KHASTKGPSVFPLAPSSRSTSESTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCDK THTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPRE EQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALH NHYTQKSLSLSPGK 325 IgG1-deltaHingeASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVICVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQK SLSLSPGK 326 IgG2-deltaHingeASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCPPC PAPPVAGPSVFLFPPKPKDTLMISRIPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTF RVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK 327 IgG2.5-deltaHingeASTKGPSVFPLAPSSRSTSESTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNIKVDKTVERKCPPC PAPPVAGPSVFLFPPKPKDTLMISRIPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTF RVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK 328 IgG1-deltaG237ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALH NHYTQKSLSLSPG 329 IgG2-plusG237ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNEGTQTYTCNVDHKPSNTKVDKTVERKSCVE CPPCPAPPVAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQ FNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK 330 IgG2.4ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCSVE CPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK 331 IgG2.3/4ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSSVE CPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK 332 IgG2.3-V13ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGDSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK 333IgG2.3-V14 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGDSVELFPPKPKDTLMISRTPEVTCVVVDVSHEDGEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK 334IgG2.3-V15 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGDSVFLFPPKPKDTLMISRTPEVTCVVVDVSDEDGEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK 335IgG2.3-V16 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGDSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDGEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPRPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK 336IgG2.3-V17 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGDSVFLFPPKPKDTLMISRTPEVTCVVVDVSDEDGEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPRPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK 337IgG2.3-V18 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK 338IgG2.3-V19 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGFPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK 339IgG2.3G1-AY-V20 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGDSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK 340IgG2.3G1-AY-V21 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGDSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDGEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK 341IgG2.3G1-AY-V22 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGDSVFLFPPKPKDTLMISRTPEVTCVVVDVSDEDGEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK 342IgG2.3G1-AY-V23 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGDSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDGEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPRPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK 343IgG2.3G1-AY-V24 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGDSVFLFPPKPKDTLMISRTPEVTCVVVDVSDEDGEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPRPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK 344IgG2.3G1-AY-V25 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGDDSVFLFPPKPKDTLMISRTPEVTCVVVDVSDEDGEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPRPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK 345IgG2.3G1-AY-V26 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPDLLGDDSVFLFPPKPKDTLMISRTPEVTCVVVDVSDEDGEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPRPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK 346IgG2.3G1-AY-V27 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVRHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK 347IgG2.3G1-AY-V28 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAPPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK 348Alternative hinge ERKCCVECPPCPAPPVAG 349 Alternative hingeERKSCVECPPCPAPPVAG 350 Alternative hinge ERKCSVECPPCPAPPVAG 351Alternative hinge ERKXCVECPPCPAPPVAG 352 Alternative hingeERKCXVECPPCPAPPVAG 353 Alternative hinge ERKCCVECPPCPAPPVAGX 354Alternative hinge ERKSCVECPPCPAPPVAGX 355 Alternative hingeERKCSVECPPCPAPPVAGX 356 Alternative hinge ERKXCVECPPCPAPPVAGX 357Alternative hinge ERKCXVECPPCPAPPVAGX 358 Alternative hingeERKCCVECPPCPAPELLGG 359 Alternative hinge ERKSCVECPPCPAPELLGG 360Alternative hinge ERKCCSVECPPCPAPELLGG 361 Alternative hingeERKXCVECPPCPAPELLGG 362 Alternative hinge ERKCXVECPPCPAPELLGG 363Alternative hinge ERKCCVECPPCPAPELLG 364 Alternative hingeERKSCVECPPCPAPELLG 365 Alternative hinge ERKCCSVECPPCPAPELLG 366Alternative hinge ERKXCVECPPCPAPELLG 367 Alternative hingeERKCXVECPPCPAPELLG 368 Alternative hinge ERKCCVECPPCPAP 369Alternative hinge ERKSCVECPPCPAP 370 Alternative hinge ERKCSVECPPCPAP371 Alternative hinge ERKXCVECPPCPAP 372 Alternative hingeERKCXVECPPCPAP 373 Portion of hinge PVAG 374 Portion of hinge ELLG 375Portion of hinge ELLGG 376 Portion of hinge SCDKTHT 377 Portion of hingeCCVE 378 wt IgG2 CH1 domain ASTKGPSVFPLAP C S R STS ES TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSS NF GTQTYTCNVDHKPSNTKVDKTV 379IgG2 CH1 and hinge ASTKGPSVFPLAP C S R STS ES TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSNFGTQTYTCNVDHKPSNTKVDKTV

380 Portion of hinge CPPCPAPThe Sequence Listing provides the sequences of the mature variableregions and heavy and light chains (i.e., sequences do not includesignal peptides).

1-62. (canceled)
 63. A method of decreasing adenosine levels in a tumorof a human subject, comprising administering to the subject an antibody,or antigen binding portion thereof, that binds to human CD73, whereinthe antibody, or antigen binding portion thereof, comprises a heavychain comprising CDR1, CDR2, and CDR3 sequences comprising the aminoacid sequences of SEQ ID NOs: 5, 6, and 7, respectively, and a lightchain comprising CDR1, CDR2, and CDR3 sequences comprising the aminoacid sequences of SEQ ID NOs: 13, 14, and 15, respectively.
 64. A methodof stimulating an immune response against a tumor in a human subject inneed thereof, comprising administering to the subject an effectiveamount of an antibody, or antigen binding portion thereof, that binds tohuman CD73, wherein the antibody, or antigen binding portion thereof,comprises a heavy chain comprising CDR1, CDR2, and CDR3 sequencescomprising the amino acid sequences of SEQ ID NOs: 5, 6, and 7,respectively, and a light chain comprising CDR1, CDR2, and CDR3sequences comprising the amino acid sequences of SEQ ID NOs: 13, 14, and15, respectively.
 65. A method of stimulating an immune response in ahuman subject comprising administering to the subject an antibody, orantigen binding portion thereof, that binds to CD73, to the subject,wherein the antibody, or antigen binding portion thereof, comprises aheavy chain comprising CDR1, CDR2, and CDR3 sequences comprising theamino acid sequences of SEQ ID NOs: 5, 6, and 7, respectively, and alight chain comprising CDR1, CDR2, and CDR3 sequences comprising theamino acid sequences of SEQ ID NOs: 13, 14, and 15, respectively.
 66. Amethod for inhibiting the growth of a tumor in a human subjectcomprising administering to the subject an antibody, or antigen bindingportion thereof, that binds to human CD73, wherein the antibody, orantigen binding portion thereof, comprises a heavy chain comprisingCDR1, CDR2, and CDR3 sequences comprising the amino acid sequences ofSEQ ID NOs: 5, 6, and 7, respectively, and a light chain comprisingCDR1, CDR2, and CDR3 sequences comprising the amino acid sequences ofSEQ ID NOs: 13, 14, and 15, respectively.
 67. A method of treatingcancer in a human subject, comprising administering to the subject atherapeutically effective amount of an antibody, or antigen bindingportion thereof, that binds to human CD73, to the subject, wherein theantibody, or antigen binding portion thereof, comprises a heavy chaincomprising CDR1, CDR2, and CDR3 sequences comprising the amino acidsequences of SEQ ID NOs: 5, 6, and 7, respectively, and a light chaincomprising CDR1, CDR2, and CDR3 sequences comprising the amino acidsequences of SEQ ID NOs: 13, 14, and 15, respectively.
 68. The method ofclaim 67, wherein the cancer is selected from the group consisting of:bladder cancer, breast cancer, uterine/cervical cancer, ovarian cancer,prostate cancer, testicular cancer, esophageal cancer, gastrointestinalcancer, pancreatic cancer, colorectal cancer, colon cancer, kidneycancer, head and neck cancer, lung cancer, stomach cancer, germ cellcancer, bone cancer, liver cancer, thyroid cancer, skin cancer, neoplasmof the central nervous system, lymphoma, leukemia, myeloma, sarcoma, andvirus-related cancer.
 69. The method of claim 67, further comprisingadministering to the subject one or more additional therapeutic agents.70. The method of claim 69, wherein the additional therapeutic agent isa PD-1 antagonist, a PD-L1 antagonist, a CTLA-4 antagonist, or a LAG-3antagonist.
 71. The method of claim 67, wherein the antibody, or antigenbinding portion thereof, comprises heavy and light chain variableregions comprising the amino acid sequences of SEQ ID NOs: 135 and 12,respectively.
 72. The method of claim 71, wherein the antibody, orantigen binding portion thereof, comprises a heavy chain sequencecomprising the amino acid sequence of SEQ ID NO: 133 or 189 and a lightchain sequence comprising the amino acid sequence of SEQ ID NO:
 102. 73.The method of claim 67, wherein the heavy chain is a full-length heavychain and the light chain is a full-length light chain.
 74. The methodof claim 71, wherein the heavy chain is a full-length heavy chain andthe light chain is a full-length light chain.
 75. The method of claim67, wherein the antibody, or antigen binding portion thereof, is an IgGantibody.
 76. The method of claim 72, wherein the antibody, or antigenbinding portion thereof, comprises a heavy chain consisting of the aminoacid sequence of SEQ ID NO: 133 and a light chain consisting of theamino acid sequence of SEQ ID NO:
 102. 77. The method of claim 72,wherein the antibody, or antigen binding portion thereof, comprises aheavy chain consisting of the amino acid sequence of SEQ ID NO: 189 anda light chain consisting of the amino acid sequence of SEQ ID NO: 102.78. The method of claim 67, wherein the antibody, or antigen bindingportion thereof, binds to human CD73 with a K_(D) of 0.1 nM to 10 nM, asdetermined by Surface Plasmon Resonance (SPR).
 79. The method of claim67, wherein the antibody, or antigen binding portion thereof, inhibitsthe activity of human CD73.
 80. The method of claim 67, wherein theantibody, or antigen binding portion thereof, comprises mediatesinternalization of human CD73.
 81. The method of claim 67, wherein theantibody, or antigen binding portion thereof, has reduced effectorfunction relative to a wild type IgG1 antibody.
 82. The method of claim67, wherein the antibody, or antigen binding portion thereof, binds tohuman CD73 with a K_(D) of 0.1 nM to 10 nM, as determined by SurfacePlasmon Resonance (SPR).